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Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis , shortens treatment in rodents relative to standard of care drugs.

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl- and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCEOleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

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.

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.

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.

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.

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.

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate ( ben ) and phthalate ( pad ), the uptake of phthalate ( pat ), and two branches of the β-ketoadipate pathway ( catRABC and pcaJIHGBLFR ). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes ( tpa ). Proteomic analyses demonstrated that the β-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca -encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL , which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent β-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative “genomic islands” (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.

Whole-genome sequencing, transcriptomic analyses, and metabolic reconstruction were used to investigate Gordonia sp. strain KTR9's ability to catabolize a range of compounds, including explosives and steroids. Aspects of this mycolic acid-containing actinobacterium's catabolic potential were experimentally verified and compared with those of rhodococci and mycobacteria.

Bacterial ring-hydroxylating dioxygenases are involved in the aerobic catabolism of a wide variety of aromatic compounds and thus play a critical role in the global carbon cycle. These multicomponent enzymes catalyze the NAD(P)H-dependent dihydroxylation of the aromatic ring, incorporating both atoms of dioxygen into the product (14, 21). Ring-hydroxylating dioxygenases have applications in the biodegradation of pollutants, many of which are aromatic compounds (49). In addition, due to their regio- and enantiospecificity, these enzymes are of burgeoning importance in generating synthons for the pharmaceutical and chemical industries (7, 22). Ring-hydroxylating dioxygenases typically consist of a hexameric oxygenase (ISP) of (αβ)3 configuration, a reductase (RED), and sometimes a ferredoxin. The α subunit of the ISP contains a Rieske-type Fe2S2 center and an active-site mononuclear iron. RED and the ferredoxin, if present, transfer reducing equivalents from NAD(P)H to the ISP. Due to their importance and potential applications, considerable effort has been focused on identifying the specificity determinants of these enzymes. Structural studies of naphthalene dioxygenase (NDO) (9) and biphenyl dioxygenase (BPDO) (12) indicate that the substrate-binding pocket is contained entirely within the C terminus of the ISP α subunit. To functionally evaluate specificity determinants, hybrid ISPs consisting of the α subunit of one enzyme and the β subunit of a related enzyme have been studied. Such experiments with NDO, BPDO, and related enzymes indicate that the α subunit is responsible for substrate preference (2, 5, 31, 40, 41). Directed mutagenesis and gene-shuffling approaches have further indicated that the α subunit harbors the principal determinants of substrate preference (3, 32, 36, 42, 43, 48). In each of these studies, enzyme function was evaluated solely in terms of substrate preference, in part due to the limited solubility of the substrates. Moreover, this preference was evaluated by using whole-cell biotransformation. Interestingly, some studies using purified hybrid ISPs indicate that the β subunit can influence the substrate preference (28, 33). This is consistent with structural data indicating that the β subunit interacts with the α subunit close to the active site of the enzyme (9, 12). Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) (EC 1.14.12.-) (27) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) (EC 1.14.12.10) are group II dioxygenases (37) (class IB according to the system of Batie et al. [4]) that catalyze the dihydroxylation of benzoates (Fig. ​(Fig.1).1). The α and β subunits of TADOmt2 are encoded by xylXY, respectively, and those of BADOADP1 are encoded by benAB, respectively (39). The RED components of TADOmt2 and BADOADP1 are encoded by xylZ and benC, respectively (25, 38). The ISP components of TADOmt2 (ISPTADO or αTβT) and BADOADP1 (ISPBADO or αBβB) share approximately 62% sequence identity yet transform different ranges of substituted benzoates. Thus, TADOmt2 transforms a wide range of substituted benzoates (54) and shows highest specificity for 3-methylbenazoate (20). In contrast, BADO transforms a much narrower range of substrates (53). The different specificities of these related enzymes and the solubility of their substrates allow kinetics studies to be performed with a wider range of substrate concentrations, thereby facilitating a more thorough investigation of the structural determinants of function in this important class of enzymes. FIG. 1. The reaction catalyzed by TADOmt2 and BADOADP1. These ring-hydroxylating dioxygenases initiate the catabolism of substituted benzoates, catalyzing their transformation to the corresponding cis-1,2-dihydroxycyclohexadienes. Each enzyme consists of two ... In the present study, BADOADP1 was overexpressed and purified by using approaches similar to those developed for TADOmt2. Hybrids ISPs consisting of the α subunit of one enzyme and the β subunit of the other were expressed and purified, and their respective specificities for a range of substituted benzoates were compared to those of the parent enzymes. The coupling of substrate utilization in the hybrid enzymes was also investigated. The contributions of the different subunits to the activities of these enzymes are discussed.

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis . Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19 Mtb ) transformed 3-oxo-4-cholesten-26-oate ( k cat / K m = 0.33 × 10 5 ± 0.03 × 10 5 M −1 s −1 ) and represents orthologs that activate the C 8 side chain of cholesterol. Both CasG RHA1 and FadD17 Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C 5 side chain of cholate, while FadD17 and its orthologs appear to activate the C 5 side chain of one or more cholesterol metabolites. CasI RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C 3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα- H -4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone ( k cat /K m = 2.4 × 10 5 ± 0.1 × 10 5 M −1 s −1 and 3.2 × 10 5 ± 0.3 × 10 5 M −1 s −1 , respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria . Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.

A structure-validated alignment of 35 extradiol dioxygenase sequences including two-domain and one-domain enzymes was derived. Strictly conserved residues include the metal ion ligands and several catalytically essential active site residues, as well as a number of structurally important residues that are remote from the active site. Phylogenetic analyses based on this alignment indicate that the ancestral extradiol dioxygenase was a one-domain enzyme and that the two-domain enzymes arose from a single genetic duplication event. Subsequent divergence among the two-domain dioxygenases has resulted in several families, two of which are based on substrate preference. In several cases, the two domains of a given enzyme express different phylogenies, suggesting the possibility that such enzymes arose from the recombination of genes encoding different dioxygenases. A phylogeny-based classification system for extradiol dioxygenases is proposed.

Genes encoding vanillin dehydrogenase ( vdh ) and vanillate O -demethylase ( vanAB ) were identified in Rhodococcus jostii RHA1 using gene disruption and enzyme activities. During growth on vanillin or vanillate, vanA was highly upregulated while vdh was not. This study contributes to our understanding of lignin degradation by RHA1 and other actinomycetes.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.

The microbial degradation of biphenyl has been well studied as a potential means of remediating soils contaminated with polychlorinated biphenyls (PCBs) (46). While the production of PCBs has been banned in industrial countries due to the adverse health effects that they cause in humans, these toxic pollutants are persistent and remain widespread in the environment (13). PCBs are aerobically transformed by the bph pathway, a pathway comprising four enzymes that initiates the catabolism of biphenyl. In most bacterial strains characterized to date, the pathway transforms up to tetrachlorobiphenyls, although some pathways can transform congeners containing up to six chlorine substituents (8, 44). A critical step in improving the microbial catabolic activities for the degradation of PCBs is understanding the reactivity of the four enzymes of the bph pathway for PCB metabolites. Biphenyl dioxygenase (BPDO), the first enzyme of the bph pathway, is a typical three-component, ring-hydroxylating dioxygenase that catalyzes the insertion of molecular oxygen into an aromatic ring, forming cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene (Fig. ​(Fig.1)1) (46). The oxygenase (BphAE) has an α3β3 composition. Each α subunit (BphA) contains a Rieske-type Fe2S2 cluster and a mononuclear iron center, located at the enzyme's active site. A reductase (BphG) and a ferredoxin (BphF) function to transfer electrons from NADH to the Rieske center of BphAE, where they are used in the hydroxylation of the biphenyl at the mononuclear iron center. The mechanism of dihydroxylation is thought to be very similar to that of the naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4, the best-characterized ring-hydroxylating dioxygenase (35). BPDO is a major determinant of a bacterium's PCB-transforming capabilities. Studies on BPDOs from different organisms have revealed significant differences in congener (substrate) preference and regiospecificity. For example, BPDOKF707 and BPDOLB00, from Pseudomonas alcaligenes KF707 and Burkholderia xenovorans LB400, respectively, show very different reactivities, although they share more than 95% sequence identity (21, 55). BPDOLB400 preferentially transforms ortho-substituted congeners containing up to six chlorines (10, 43). This enzyme has the relatively unusual ability to catalyze the 3,4-dihydroxylation of certain 2,5-substituted congeners, such as 2,2′,5,5′-tetrachlorobiphenyl (32). BPDOLB400 is also remarkable in that it catalyzes the dehalogenation of certain 2-Cl congeners, yielding 2,3-dihydroxybiphenyls (25, 50-52), the substrate of the third Bph pathway enzyme. By contrast, BPDOKF707 transforms a narrow range of PCB congeners and catalyzes neither 3,4-dihydroxylation nor o-dechlorination. Moreover, it preferentially transforms 4,4′-dichlorobiphenyl over either 3,3′- or 2,2′-dichlorobiphenyl (24). A third enzyme, BPDOB356 from Pandoraea pnomenusa (formerly Comamonas testosteroni [59]) B356, shares approximately 70% sequence identity with both BPDOKF707 and BPDOLB400. Previous studies have indicated this enzyme's preference for meta- > ortho- > para-substituted dichlorobiphenyls (33), as well as its higher specific activity against biphenyl than that of BPDOLB00. FIG. 1. Dihydroxylation of biphenyl by BPDO. The enzyme comprises a flavin adenine dinucleotide-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and an oxygenase that contains a Rieske-type Fe2S2 cluster and a catalytic mononuclear iron center. Biphenyl ... Protein engineering efforts have yielded BPDO with improved PCB-degrading capabilities, as well as insights into the molecular basis of congener preference. Mutagenesis studies of BphALB400 and BphAKF707 identified four regions (regions I to IV) whose sequences influence the range of congeners attacked (44). All four regions occur in the C-terminal domain of BphA, consistent with the location of the oxygenase's active site. Substitution of individual residues in region III of BphALB400, comprising Thr335, Phe336, Asn338, and Ile341, improved the ability of the enzyme to transform 4,4′-dichlorobiphenyls, although the greatest improvements in activity were achieved by multiple substitutions in this region, suggesting that there is a cooperative or additive effect (4). Several studies have since confirmed the importance of these residues in determining the enzyme's congener preference and regiospecificity (3-5, 36, 54). For example, BphAEII9, a variant of BphAELB400 containing region III of BphAB356, is reported to possess better PCB-degrading properties than either parental enzyme (3). By contrast, BphAEII10, which differs from BphAEII9 by a single residue at position 267 (Ser, as in BphAEB356, instead of Ala, as in BphAELB400), was not able to transform any of the PCBs tested. Here, we report the characterization of four variants of BPDO: BphAELB400, BphAEB356, and two variants of these enzymes generated via directed evolution, BphAEII9 and BphAEII10. The steady-state kinetic parameters for biphenyl of anaerobically purified non-His-tagged enzymes were determined, and the activities towards various PCB congeners were investigated. The degree of uncoupling between O2 utilization and congener transformation was also determined for the different enzymes using different congeners. To validate previous studies, activities determined using purified enzymes and whole cells were compared, as were activities determined using individual congeners and mixtures of congeners. Finally, a crystal structure of the BphAEB356-2,6-dichlorobiphenyl complex was determined. The results are discussed in terms of the proposed catalytic mechanism of ring-hydroxylating dioxygenases and the specificities of the different BPDOs.

In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN , encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF , encoding phenylacetyl-CoA ligase, and paaR , encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l -phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria.

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBD LB400 ; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-dichloro-2,3-dihydroxybiphenyl (2′,6′-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBD P6 -I and DHBD P6 -III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBD LB400 , both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBD P6 -I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBD P6 -I were among the best for DHBD P6 -III (4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB; DHBD P6 -III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBD P6 -I and 2′-Cl DHB for DHBD P6 -III. As observed in DHBD LB400 , chloro substituents influenced the reactivity of the dioxygenases with O 2 . For example, the apparent specificities of DHBD P6 -I and DHBD P6 -III for O 2 in the presence of 2′-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBD P6 -I and DHBD P6 -III shared the relative inability of DHBD LB400 to cleave 2′,6′-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s −1 , respectively). However, these isozymes had remarkably different apparent K m values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 μM for DHBD LB400 , DHBD P6 -I, and DHBD P6 -III, respectively). The markedly different reactivities of DHBD P6 -I and DHBD P6 -III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701–15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD P6 , was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD P6 hydrolyzed HOPDA with a k cat / K m of 1.62 (± 0.03) × 10 7 M −1 s −1 (100 mM phosphate [pH 7.5], 25°C), which is within 70% of that of BphD LB400 . BphD P6 was also similar to BphD LB400 in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD P6 more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD LB400 . These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.