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Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the Δ fcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C 17 versus C 16 ). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s−1 and 2.5 ± 0.2 × 107 m−1 s−1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C–C–hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

In the catabolism of lignin-derived biphenyl by Sphingobium sp. SYK-6, LigZ catalyzes the cleavage of 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) to a meta-cleavage product (MCP) identified here as 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA). DCHM-HOPDA is transformed nonenzymatically, likely to a lactone (k = 0.13 ± 0.01 min-1 , pH 7.5). This is hydrolyzed to the dienolate at alkaline pH (apparent pKa ~ 11.3). Only the dienolate is a substrate for LigY, the putative MCP hydrolase. LigZ has higher specificity for OH-DDVA (kcat /Km = 2.20 ± 0.02 × 107 s-1 ·m-1 ) than for protocatechuate (PCA; 6 ± 1 × 102 s-1 ·m-1 ). PCA also inactivates LigZ (partition ratio of 50), but at rates too low to be physiologically relevant. This study provides insight into the bacterial catabolism of lignin and facilitates the study of downstream catabolic enzymes.

The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac’s substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes.

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >10(5) s(-1) M(-1) for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (Ki S ∼100 μM). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue "mouth loop," which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 A in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a major soil and groundwater contaminant. Organisms such as Gordonia sp. KTR9, capable of degrading RDX and using it as an N source, may prove useful for bioremediation of contaminated sites. XplA is a cytochrome P450 monooxygenase responsible for RDX degradation. Expression of xplA in KTR9 was not induced by RDX but was strongly induced (50-fold) during N-limited growth. When glnR, encoding a regulatory protein affecting N assimilation in diverse Actinobacteria, was deleted from KTR9, the bacterium lost the ability to use nitrate, nitrite, and RDX as N sources. Deletion of glnR also abolished the inhibition of xplA expression by nitrite. Our results confirm the essential role of GlnR in regulating assimilation of nitrite, but there was no evidence for a direct role of GlnR in regulating XplA expression. Rather, the general availability of nitrogen repressed XplA expression. We conclude that the inability of the glnR mutant to use RDX as an N source was due to its inability to assimilate nitrite, an intermediate in the assimilation of nitrogen from RDX. Regulation of XplA does not seem adaptive for KTR9, but it is important for RDX bioremediation with KTR9 or similar bacteria.

Summary Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ1/4-3-ketocholate and Δ1,4-3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3′propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ1/4-3-ketocholate, and Δ1,4-3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

Summary Cholesterol catabolism is widespread in actinobacteria and is critical for Mycobacterium tuberculosis (Mtb) virulence. Catabolism of steroid nucleus rings C and D is poorly understood: it is initiated by the CoA thioesterification of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) by FadD3, whose gene is part of the KstR2 regulon. In Mtb, genes of this regulon were upregulated up to 30- and 22-fold during growth on cholesterol and HIP, respectively, versus another minimal medium. In contrast, genes involved in degrading the cholesterol side-chain and nucleus rings A and B were only upregulated during growth on cholesterol. Similar results were obtained in Rhodococcus jostii RHA1. Moreover, the regulon was not upregulated in a ΔfadD3 mutant unable to produce HIP-CoA. In electrophoretic mobility shift assays, HIP-CoA relieved the binding of KstR2Mtb to each of three KstR2 boxes: CoASH, HIP and a related CoA thioester did not. Inspection of the structure of KstR2RHA1 revealed no obvious HIP-CoA binding pocket. The results establish that Mtb can catabolize the entire cholesterol molecule and that HIP-CoA is an effector of KstR2. They further indicate that KstR2 specifically represses the expression of the HIP degradation genes in actinobacteria, which encode a lower pathway involved in the catabolism of multiple steroids.

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/β-hydrolases.

The aromatic polymer lignin represents a possible renewable source of aromatic chemicals, if biocatalytic routes for lignin breakdown can be developed. The availability of a genome sequence for Rhodococcus jostii RHA1, a bacterium that breaks down lignin, has allowed the application of a targeted pathway engineering strategy to lignin breakdown to produce vanillin, a valuable food/flavor chemical. A gene deletion strain of R. jostii RHA1 in which the vanillin dehydrogenase gene had been deleted, when grown on minimal medium containing 2.5% wheat straw lignocellulose and 0.05% glucose, was found to accumulate vanillin with yields of up to 96 mg/L after 144 h, together with smaller amounts of ferulic acid and 4-hydroxybenzaldehyde.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10−9 m) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 102–104 times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn(2+)), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme's apparent k(cat) and k(cat)/K(m) values for Mn(2+) by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn(2+) to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn(2+)-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant's high K(m) for Mn(2+) (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.

The bacterial uptake of steroids and their metabolites remains poorly understood. We investigated two transporters associated with cholate catabolism in Rhodococcus jostii RHA1. Reverse transcriptase quantitative-PCR indicated that an ATP-binding cassette (ABC) transporter and a major facilitator superfamily (MFS) transporter were upregulated 16.7- and 174-fold, respectively, during the exponential phase of growth on cholate compared to growth on pyruvate. Gene knockout analysis established that these transporters are required for the reassimilation of distinct metabolites that accumulate during growth on cholate. The ABC transporter, encoded by camABCD , was essential for uptake of 1β(2′-propanoate)-3aα- H -4α(3″ (R) -hydroxy-3″-propanoate)-7aβ-methylhexahydro-5-indanone and a desaturated analog. The MFS transporter, encoded by camM , was essential for uptake of 3,7 (R) ,12 (S) -trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oate. These metabolites differ from cholate metabolites reported to be excreted by proteobacteria in that they retain an isopropanoyl side chain at C-17. The uptake of these metabolites was necessary for maximal growth on cholate: a Δ camB mutant lacking the permease component of the ABC transporter and a Δ camM mutant lacking the MFS transporter grew to 74% and 77%, respectively, of the yield of the wild type. This study demonstrates for the first time the requirement for specific transporters for uptake of cholate metabolites and highlights the importance and complexity of transport processes associated with bacterial steroid catabolism.

The cholesterol catabolic pathway occurs in most mycolic acid-containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3(Mtb) catalysed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α-OH HIP and 1β-OH HIP. The apparent specificity constant (k(cat) /K(m) ) of FadD3(Mtb) for HIP was 7.3 ± 0.3 × 10(5) M(-1) s(-1) , 165 times higher than for 5α-OH HIP, while the apparent K(m) for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3(Mtb) did not detectably transform a metabolite with a partially degraded C17 side-chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side-chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.

Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAE II9 , a variant of BphAE LB400 in which a seven-residue segment, 335 TFNNIRI 341 , has been replaced by the corresponding segment of BphAE B356 , 333 GINTIRT 339 , transforms a broader range of PCB congeners than does either BphAE LB400 or BphAE B356 , including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAE II9 , we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAE LB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAE II9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAE II9 transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAE LB400 and BphAE B356 . BphAE II9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent k cat / K m of 2.2 ± 0.5 mM −1 s −1 , versus 0.9 ± 0.5 mM −1 s −1 for BphAE B356 ). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes. IMPORTANCE Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.

Meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.

DypB from Rhodococcus jostii RHA1 is a bacterial dye-decolorizing peroxidase (DyP) that oxidizes lignin and Mn(II). Three residues interact with the iron-bound solvent species in ferric DypB: Asn-246 and the conserved Asp-153 and Arg-244. Substitution of either Asp-153 or Asn-246 with alanine minimally affected the second order rate constant for Compound I formation (k1 ∼ 105 m−1s−1) and the specificity constant (kcat/Km) for H2O2. Even in the D153A/N246A double variant, these values were reduced less than 30-fold. However, these substitutions dramatically reduced the stability of Compound I (t½ ∼ 0.13 s) as compared with the wild-type enzyme (540 s). By contrast, substitution of Arg-244 with leucine abolished the peroxidase activity, and heme iron of the variant showed a pH-dependent transition from high spin (pH 5) to low spin (pH 8.5). Two variants were designed to mimic the plant peroxidase active site: D153H, which was more than an order of magnitude less reactive with H2O2, and N246H, which had no detectable peroxidase activity. X-ray crystallographic studies revealed that structural changes in the variants are confined to the distal heme environment. The data establish an essential role for Arg-244 in Compound I formation in DypB, possibly through charge stabilization and proton transfer. The principle roles of Asp-153 and Asn-246 appear to be in modulating the subsequent reactivity of Compound I. These results expand the range of residues known to catalyze Compound I formation in heme peroxidases.

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K(m)) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K(m)(O(2)) was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.

Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis , arguably the world’s most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.

Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain Gordonia sp. KTR9. This plasmid carries xplA , encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with xplA are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator. Rhodococcus jostii RHA1 expressing xplA from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an Escherichia coli strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of glnA-xplB , xplA , and xplR showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3C(pro), prefers Ile, Val, or Leu at the P(4) position of the cleavage sequence and Gly, Ser, or Ala at the P'(1) position. The assay can also be used to screen small-molecule libraries for inhibitors.

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (k(cat)/K(m) = 3.3 +/- 0.3 x 10(4) m(-1) s(-1)) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K(d) of 51 +/- 2 mum. The S114A.DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8-1.9 Aindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the alpha-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.

Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by Rhodococcus jostii RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 +/- 0.02 microM and 0.27 +/- 0.05 microm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9alpha-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.

KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance.

Bioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen with its human host. Here, we provide direct evidence that the Mce4 system of Rhodococcus jostii RHA1 is a steroid uptake system. Transcriptional analyses indicate that the system is encoded by an 11-gene operon, up-regulated 4.0-fold during growth on cholesterol versus on pyruvate. Growth of RHA1 on cholesterol and uptake of radiolabeled cholesterol both required expression of genes in the mce4 operon encoding two permeases plus eight additional proteins of unknown function. Cholesterol uptake was ATP-dependent and exhibited Michaelis-Menten kinetics with a K(m) of 0.6 +/- 0.1 mu M. This uptake system was also essential for growth of RHA1 on beta-sitosterol, 5-alpha-cholestanol, and 5-alpha-cholestanone. Bioinformatic analysis revealed that all mce4 loci in sequenced genomes are linked to steroid metabolism genes. Thus, we predict that all Mce4 systems are steroid transporters. The transport function of the Mce4 system is consistent with proposed roles of cholesterol and its metabolism in the pathogenesis of M. tuberculosis.

The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.

The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

Graphical abstract A series of ketones and fluorinated ketones have been synthesized as potent reversible inhibitors of SARS 3CLpro. Three aromatic rings, including a 3-bromopyridin-5-yl moiety, enhanced inhibition of this enzyme., The severe acute respiratory syndrome (SARS) virus depends on a chymotrypsin-like cysteine proteinase (3CLpro) to process the translated polyproteins to functional viral proteins. This enzyme is a target for the design of potential anti-SARS drugs. A series of ketones and corresponding mono- and di-fluoro ketones having two or three aromatic rings were synthesized as possible reversible inhibitors of SARS 3CLpro. The design was based on previously established potent inhibition of the enzyme by oxa analogues (esters), which also act as substrates. Structure–activity relationships and modeling studies indicate that three aromatic rings, including a 5-bromopyridin-3-yl moiety, are key features for good inhibition of SARS 3CLpro. Compound 11d, 2-(5-bromopyridin-3-yl)-1-(5-(4-chlorophenyl)furan-2-yl)ethanone and its α-monofluorinated analogue 12d, gave the best reversible inhibition with IC50 values of 13 μM and 28 μM, respectively. In contrast to inhibitors having two aromatic rings, α-fluorination of compounds with three rings unexpectedly decreased the inhibitory activity.

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.

Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.

The microbial degradation of nitriles is of interest for bioremediation and green chemistry. We demonstrated that the soil bacterium Rhodococcus sp. RHA1 utilizes a range of nitriles, including acetonitrile, as growth substrates. Proteomic analysis identified 13 proteins that were more abundant in acetonitrile-grown cells, including an aliphatic amidase and a protein with no known homologue. Purification of a nitrile hydratase (NHase) from acetonitrile-grown cells identified the unknown protein as the beta subunit of a two-subunit NHase. Sequence analysis revealed that the genes encoding the amidase (anhC) and the NHase (anhAB) occur in a 12.8 kbp cluster located on plasmid pRHL2. The anh gene cluster also encodes an acetyl-CoA hydrolase, transcriptional regulators, a putative cobalt transporter and a protein of unknown function. Striking features of the NHase include the amino acid sequence identity (32%) and large size (63 and 56 kDa) of the alpha and beta subunits, as well as the enzyme's metal ion content (one cobalt, two copper and one zinc). The enzyme possessed similar specificities for acetonitrile and propionitrile (k(cat)/K(m) approximately 7 mM(-1) s(-1)) followed by acrylonitrile and butyronitrile. We propose that this acetonitrile hydratase (ANHase) represents the first member of a previously unknown class of NHases.

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (λmax is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (λmax is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (λmax is 506 nm) with a similar rate, 1/τ ∼ 500 s-1. The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7A away.

The 3C-like main peptidase 3CL(pro) is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL(pro) and the structurally related 3C(pro) viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL(pro) in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.

Chlorinated aromatic compounds, such as polychlorinated biphenyls (PCBs) and dibenzofurans, are among the most widespread, toxic, and/or persistent environmental pollutants. The Bph and Dxn/Dbf pathways responsible for the aerobic bacterial catabolism of biphenyl and dibenzofuran, respectively, have been extensively studied, in part due to their potential for remediating environments contaminated with the polychlorinated compounds (2, 10). The pathways share three homologous enzymes (Fig. ​(Fig.1):1): a multicomponent dioxygenase that catalyzes the initial step of ring hydroxylation, an extradiol dioxygenase that catalyzes oxygenolytic cleavage of the catecholic intermediate at the meta position to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) or ortho-substituted HOPDA, and a meta-cleavage product (MCP) hydrolase that catalyzes an unusual C—C bond cleavage of the HOPDA. FIG. 1. Microbial catabolism of dibenzofuran and biphenyl by the Dbf and Bph pathways, respectively. Carbon atoms 1, 6, and 8 of 8-OH HOPDA are numbered. The Dbf pathway enzymes are as follows: DxnA1 and DxnA2, large and small subunits of dioxin dioxygenase, ... A general limitation of bacterial catabolic pathways for the degradation of complex industrial mixtures is that one or more pathway enzymes lack the requisite broad substrate specificity to degrade all man-made (xenobiotic) congeners of the naturally occurring compounds, and some enzymes are susceptible to inhibition by the chlorinated metabolites. Studies have identified MCP hydrolases as a bottleneck in the Bph pathway, as summarized below, as well as in the toluene catabolic pathway (11). Substrate specificity studies of MCP hydrolases from potent PCB degraders, BphDLB400 from Burkholderia xenovorans LB400 and BphDP6 from Rhodococcus globerulus P6, revealed that these enzymes are inefficient in transforming HOPDA chlorinated at the 3 or 4 position (30, 31). In addition, these poorly transformed chlorinated metabolites are potential competitive inhibitors of the enzymes, thereby reducing the efficiency of transformation of all congeners, including unsubstituted HOPDA. Consistent with these studies, HOPDAs accumulate during the bacterial degradation of some PCBs (12). Interest in the inhibition of MCP hydrolases is further heightened with the recent discovery of HsaDH37Rv of Mycobacterium tuberculosis H37Rv (35). Formerly annotated as BphD, HsaDH37Rv appears to be involved in steroid metabolism during the survival of M. tuberculosis in the macrophage, hydrolyzing 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA). MCP hydrolases are members of the α/β-hydrolase superfamily (28). The hydrolyzed C—C bond lies between a dienoate (C-1 to C-5) (Fig. ​(Fig.1)1) and a carbonyl (C-6). Hydrolysis is mediated by conserved Ser-His-Asp residues reminiscent of the catalytic triad of proteases. The proposed catalytic mechanism involves His-mediated ketonization of the dienoate (18, 23), followed by Ser-mediated hydrolysis at C-6. It is unclear whether the catalytic Ser attacks C-6 directly to form an acyl-enzyme intermediate or whether it activates a water molecule which then attacks C-6 (9, 18, 23, 24). Crystal structures have been reported for five MCP hydrolases: BphDLB400 (18), BphDRHA1 from Rhodococcus sp. strain RHA1 (26), CumDIP01 from Pseudomonas fluorescens IP01, involved in cumene degradation (13), CarCJ3 from Janthinobacterium sp. strain J3, involved in carbazole degradation (14), and MhpC (7). All comprise a “core domain,” consisting of an eight-stranded β-sheet flanked by α-helices, and a “lid domain,” which occurs as an insertion into the core domain. The active site, located between the core and lid domains, includes the conserved Ser, His, and Asp residues and is divided into two subsites: P (polar) and NP (nonpolar), located on each side of the catalytic serine. The P subsite binds the dienoate moiety, and the NP subsite accommodates the C-6 substituent, a phenyl ring in the case of HOPDA. Sphingomonas wittichii RW1, one of the best-characterized dibenzofuran-degrading bacteria (37), is able to grow on either dibenzofuran or dibenzo-p-dioxin as a sole organic substrate (36). In addition, the strain partially transforms 2-Cl, 3-Cl, 4-Cl, and 2,3-diCl dibenzofurans to the corresponding chlorinated hydroxysalicylates (36). It is not entirely clear which enzymes are responsible for these transformations, as the bacterium possesses multiple homologues of key catabolic enzymes. For example, Armengaud and coworkers isolated four genes that encode α subunits of ring-hydroxylating dioxygenases and two genes that encode β subunits (2). The bacterium also contains at least two extradiol dioxygenases and three MCP hydrolases (2, 4). Two of the MCP hydrolases, DxnB and DxnB2 (previously H1 and H2), were purified and shown to hydrolyze HOPDA and 8-hydroxy HOPDA (4). In addition, the gene encoding a third potential MCP hydrolase was cloned (2). DxnB2 has been reported to be more related to MCP hydrolases involved in the catabolism of monocyclic aromatic compounds than to enzymes such as BphD, which are involved in the degradation of bicyclic aromatics (15). This result, derived from amino acid sequence comparisons, is somewhat surprising as the substrates of DxnB and BphD differ by a single hydroxyl group on the phenyl ring. Better characterization of the DxnB enzymes may clarify the phylogenetic relationships of MCP hydrolases and provide insight into why S. wittichii RW1 contains multiple isozymes. Herein, we report the cloning, sequencing, and heterologous expression of dxnB2. Heterologously produced DxnB2 was purified, and steady-state kinetic studies revealed that the enzyme possesses useful activities towards chlorinated HOPDAs. We generated a structure-based sequence alignment of MCP hydrolases and used this to perform phylogenetic analyses. The results identified a previously unrecognized class of MCP hydrolases and revealed possible structural determinants of substrate specificity in the various classes.

Phthalate isomers and their esters are important pollutants whose biodegradation is not well understood. Rhodococcus sp. strain RHA1 is notable for its ability to degrade a wide range of aromatic compounds. RHA1 was previously shown to degrade phthalate (PTH) and to have genes putatively encoding terephthalate (TPA) degradation. Transcriptomic analysis of 8,213 genes indicated that 150 were up-regulated during growth on PTH and that 521 were up-regulated during growth on TPA. Distinct ring cleavage dioxygenase systems were differentially expressed during growth on PTH and TPA. Genes encoding the protocatechuate (PCA) pathway were induced on both substrates, while genes encoding the catechol branch of the PCA pathway were additionally induced only on TPA. Accordingly, protocatechuate-3,4-dioxygenase activity was induced in cells grown on both substrates, while catechol-1,2-dioxygenase activity was induced only in cells grown on TPA. Knockout analysis indicated that pcaL , encoding 3-oxoadipate enol-lactone hydrolase and 4-carboxymuconolactone decarboxylase, was required for growth on both substrates but that pcaB , encoding β-carboxy- cis , cis -muconate lactonizing enzyme, was required for growth on PTH only. These results indicate that PTH is degraded solely via the PCA pathway, whereas TPA is degraded via a bifurcated pathway that additionally includes the catechol branch of the PCA pathway.

The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.

Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis , degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC , supAB , and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC − mutant accumulated 3,4-DHSA, indicating that HsaC RHA1 , formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette–Guérin. M. bovis bacillus Calmette–Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC H37Rv and HsaD H37Rv transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities ≈40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis 's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes have wide catalytic versatility but are rarely known for their ability to react with oxygen to catalyze challenging reactions. Here, using in vitro reconstitution and kinetic analysis, we report that the indolmycin biosynthetic enzyme Ind4, from Streptomyces griseus ATCC 12648, is an unprecedented O2- and PLP-dependent enzyme that carries out a four-electron oxidation of L-arginine, including oxidation of an unactivated carbon-carbon (C-C) bond. We show that the conjugated product of this reaction, which is susceptible to nonenzymatic deamination, is efficiently intercepted and stereospecifically reduced by the partner enzyme Ind5 to give D-4,5-dehydroarginine. Thus, Ind4 couples the redox potential of O2 with the ability of PLP to stabilize anions to efficiently oxidize an unactivated C-C bond, with the subsequent stereochemical inversion by Ind5 preventing off-pathway reactions. Altogether, these results expand our knowledge of the catalytic versatility of PLP-dependent enzymes and enrich the toolbox for oxidative biocatalysis.

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol., Author Summary Human beings are the sole ecological niche for M. tuberculosis (Mtb), and it is estimated that 1.8 billion people are currently infected with Mtb. An important aspect of this infection is Mtb’s ability to maintain infection by replicating within macrophages. Within macrophages, Mtb exploits a specialized set of metabolic pathways to utilize host-derived nutrients, such as fatty acids and/or cholesterol, for energy production. Many details regarding Mtb metabolism during infection remain unknown. Here we took a chemical approach to identify small molecule probes, which target Mtb metabolism during infection in macrophages. We found that many of the small molecule inhibitors that we identified require cholesterol for activity. Here we report a novel chemical rescue approach to identify the metabolic targets of three novel inhibitors, and discovered that cAMP signaling is linked to cholesterol utilization in Mtb. Together, these data demonstrate that cholesterol exerts a dominant effect on Mtb metabolism within macrophages. Additionally, the novel inhibitors identified in this study will facilitate evaluation of cholesterol metabolism as a target for chemotherapeutic intervention.

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds ( 30 ) or steroids ( 4 ). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O 2 -rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe 3 S 4 ] cluster. This conclusion was corroborated by UV-Visible absorption ( e 412 = 13.4 mM − 1 cm − 1 ) and EPR ( g x,y = 2.00 and g z = 2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was − 177.0 ± 0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 °C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.

Extradiol dioxygenases play a key role in determining the specificities of the microbial aromatic catabolic pathways in which they occur. To identify the structural determinants of specificity in this class of enzymes, variants of 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD) were investigated. Structural data of the DHBD/DHB complex informed the design of seven variants at four positions: V148W, V148L, M175W, A200I, A200W, P280W, and V148L/A200I. All variants had reduced specificity for DHB. In addition, the V148W, V148L, A200I, and V148L/A200I variants had increased specificity for catechol. Indeed, the V148W variant had a higher apparent specificity for 3-Me catechol than for DHB, although the substitution reduced the kcat for all tested substrates as well as the rate constant of suicide inactivation of the enzyme. These results are consistent with available structural data which suggest that the larger residue at position 148 may partially occlude O2 binding. The results further indicate that in addition to defining substrate specificity, the binding pocket orientates the bound catechol to minimize oxidative inactivation of the enzyme during catalysis.

DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of > 10 5 colonies/h. The best variant, designated DoxG S M A 2 , cleaved 3,4-DHB with an apparent specificity constant of 2.0 ′ 0.3 × 10 6 M - 1 s - 1 , which is 770 times that of wild-type (WT) DoxG. The specificities of DoxG S M A 2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxG S M A 2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.

The main peptidase (M(pro)) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive molecular targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV M(pro) by an aza-peptide epoxide (APE; k(inact)/K(i) = 1900(+/-400) M(-1) s(-1)). The crystal structures of the M(pro):APE complex in the space groups C2 and P2(1)2(1)2(1) revealed the formation of a covalent bond between the catalytic Cys145 S(gamma) atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of M(pro) in a substrate-like manner, with excellent structural and chemical complementarity. In addition, the crystal structure of unbound M(pro) in the space group C2 revealed that the "N-fingers" (N-terminal residues 1 to 7) of both protomers of M(pro) are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystallization pH of 6.5, contrary to the previously determined crystal structures of unbound M(pro) in the space group P2(1).