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Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon–carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C(17)–C(20) bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon–carbon bond scission.

D-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of D-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D(2)O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade ...

In the biosynthesis of the macrolactam antibiotic cremimycin, the 3-aminononanoic acid starter unit is formed via a non-2-enoyl acyl carrier protein thioester intermediate, which is presumed to be constructed by cis-acyltransferase (AT) polyketide synthases (PKSs) CmiP2, CmiP3, and CmiP4. While canonical cis-AT PKS modules are comprised of a single polypeptide, the PKS module formed by CmiP2 and CmiP3 is split within the dehydratase (DH) domain. Here, we report the enzymatic function and the structural features of this split-DH domain. In vitro analysis showed that the split-DH domain catalyzes the dehydration reaction of (R)-3-hydroxynonanoyl N-acetylcysteamine thioester (SNAC) to form (E)-non-2-enoyl-SNAC, suggesting that the split-DH domain is catalytically active in cremimycin biosynthesis. In addition, structural analysis revealed that the CmiP2 and CmiP3 subunits of the split-DH domain form a tightly associated heterodimer through several hydrogen bonding and hydrophobic interactions, which are similar to those of canonical DH domains of other cis-AT PKSs. These results indicate that the split-DH domain has the same function and structure as common cis-AT PKS DH domains.

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent kcat of 3.0 s-1. The product, l-glycero-l-galacto-octuluronate-1-phosphate, has a kcat/Km value of 2.1 × 103 M-1 s-1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l-glycero-l-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 A (Protein Data Bank entry 6OFU ), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l-glycero-l-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l-glycero-l-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, β-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and β-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 μg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring β-thujaplicin and the α- and β-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 μM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

Sulfoxides and sulfones are commonly found in nature as a result of thioether oxidation, whereas only a very few enzymes have been found to metabolize these compounds. Utilizing the strong reduction potential of the [4Fe-4S] cluster of radical S-adenosyl-l-methionine (SAM) enzymes, we herein report the first enzyme-catalyzed reductive cleavage of sulfoxide and sulfone. We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM. NosL cleaves all of the three bonds (i.e., S–C(5′), S–C(γ), and S–O) connecting the sulfur center of SAHO, with a preference for S–C(5′) bond cleavage. Similar S–C cleavage activity was also found for SHAO2, but no S–O cleavage was observed. In contrast to NosL, NosN almost exclusively cleaves the S–C(5′) bonds of SAHO and SAHO2 with much higher efficiencies. Our study provides valuable insights into the [4Fe-4S] cluster-mediated red...

Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO(2) and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO(2) binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn(2+) coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein–ligand recognition. Through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.

Linear triquinanes are sesquiterpene natural products with hydrocarbon skeletons consisting of three fused 5-membered rings. Importantly, several of these compounds exhibit useful anti-cancer, anti-inflammatory, and antibiotic properties. However, linear triquinanes pose significant challenges to organic synthesis due to the structural and stereochemical complexity of their hydrocarbon skeletons. To illuminate nature’s solution to the generation of linear triquinanes, we now describe the crystal structure of Streptomyces clavuligerus cucumene synthase. This sesquiterpene cyclase catalyzes the stereospecific cyclization of farnesyl diphosphate to form a linear triquinane product, (5S,7S,10R,11S)-cucumene. Specifically, we report the structure of the wild-type enzyme at 3.05 Å resolution and the structure of the T181N variant at 1.96 Å resolution, both in the open active site conformations without any bound ligands. The high-resolution structure of T181N cucumene synthase enables inspection of the active site contour, which adopts a three-dimensional shape complementary to a linear triquinane. Several aromatic residues outline the active site contour and are believed to facilitate cation-π interactions that would stabilize carbocation intermediates in catalysis. Thus, aromatic residues in the active site not only define the template for catalysis, but these residues also play a role in reducing activation barriers in the multi-step cyclization cascade.

DNA polymerase θ (Pol θ) is a multifunctional enzyme with double-strand break (DSB) repair, translesion synthesis, and lyase activities. Pol θ lyase activity on ternary substrates containing a 5'-dRP that are produced during base excision repair of abasic sites (AP) is weak compared to that of DNA polymerase β (Pol β), a polymerase integrally involved in base excision repair. This led us to explore whether Pol θ utilizes its lyase activity to remove 5'-dRP and incise abasic sites from alternative substrates that might be produced during DNA damage and repair. We found that Pol θ exhibited lyase activity on abasic lesions near DSB termini and on clustered lesions. To calibrate the Pol θ activity, Pol β reactivity was examined with the same substrates. Pol β excised 5'-dRP from within a 5'-overhang 80 times faster than did Pol θ. Pol θ and Pol β also incised AP within clustered lesions but showed opposite preferences with respect to the polarity of the lesions. AP lesions in 5'-overhangs were typically excised by Pol β 35-50 times faster than those in a duplex substrate but 15-20-fold more slowly than 5'-dRP in a ternary complex. This is the first report of Pol θ exhibiting lyase activity within an unincised strand. These results suggest that bifunctional polymerases may exhibit lyase activity on a greater variety of substrates than previously recognized. A role in DSB repair could potentially be beneficial, while the aberrant activity exhibited on clustered lesions may be deleterious because of their conversion to DSBs.

Tyrosine phenol-lyase (TPL; EC 4.1.99.2) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-tyrosine to phenol and ammonium pyruvate. We have shown previously that F448A TPL has kcat and kcat/Km values for l-tyrosine reduced by ∼104-fold [Phillips, R. S., Vita, A., Spivey, J. B., Rudloff, A. P., Driscoll, M. D., and Hay, S. (2016) ACS Catal. 6, 6770–6779]. We have now obtained crystal structures of F448A TPL and complexes with l-alanine, l-methionine, l-phenylalanine, and 3-F-l-tyrosine at 2.05–2.27 A and the complex of wild-type TPL with l-phenylalanine at 1.8 A. The small domain of F448A TPL, where Phe-448 is located, is more disordered in chain A than in wild-type TPL. The complexes of F448A TPL with l-alanine and l-phenylalanine are in an open conformation in both chains, while the complex with l-methionine is a 52:48 open:closed equilibrium mixture in chain A. Wild-type TPL with l-alanine is closed in chain A and open in chain B, and the complex with l...

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues - E64, S73, S75, and Y192 - as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner–Doudoroff pathway and a branched Entner–Doudoroff pathway, respecti...

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.

Marine organisms release dimethylsulfide (DMS) via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known, yet these exhibit highly variable levels of activity. Most assigned bacterial DMSP lyases, including DddK, DddL, DddQ, DddW, and DddY, appear to belong to one, cupin-like superfamily. Here, we attempted to define and map this superfamily dubbed cupin-DLL (DMSP lyases and lyase-like). To this end, we have pursued the characterization of various recombinant DMSP lyases belonging to this superfamily of metalloenzymes, and especially of DddY and DddL that seem to be the most active DMSP lyases in this superfamily. We identified two conserved sequence motifs that characterize this superfamily. These motifs include the metal-ligating residues that are absolutely essential and other residues including an active site tyrosine that seems to play a relatively minor role in DMSP lysis. We also identified a transition metal chelator, N, N, N', N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), that selectively inhibits all known members of the cupin-DLL superfamily that exhibit DMSP lyase activity. A phylogenetic analysis indicated that the known DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily multiple times. However, unusually low specific DMSP lyase activity and genome context analysis suggest that DMSP lyase is not the native function of most cupin-DLL families. Indeed, a systematic profiling of substrate selectivity with a series of DMSP analogues indicated that some members, most distinctly DddY and DddL, are bona fide DMSP lyases, while others, foremost DddQ, may only exhibit promiscuous DMSP lyase activity.

DesII is a radical SAM lyase that catalyzes a deamination reaction during the biosynthesis of desosamine in Streptomyces venezuelae. Competing mechanistic hypotheses for this radical-mediated reaction are differentiated according to whether or not a 1, 2-migration takes place and the timing of proton abstraction following generation of a substrate α-hydroxyalkyl radical intermediate. In the present study, the deuterated C4-epimer of the natural substrate, TDP-4-amino-4-deoxy-d-[3-2H]fucose, was prepared and shown to be a substrate for DesII undergoing deamination alone with a specific activity that is only marginally reduced (ca. 3-fold) with respect to deamination of the natural substrate. Furthermore, pH titration of the deamination reaction implicates the presence of a hydron acceptor that facilitates catalysis but does not appear to be necessary. Based on these as well as previously reported results, a mechanism is proposed involving direct elimination of ammonium concerted with proton transfer to the nucleofuge from the adjacent α-hydroxyalkyl radical.

The natural aminocarboxylic acid product ethylenediamine- N, N'-disuccinic acid [( S, S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediaminetetraacetic acid (EDTA), which is currently used on a large scale in numerous applications. Previous studies have demonstrated that biodegradation of ( S, S)-EDDS may be initiated by an EDDS lyase, converting ( S, S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited because of the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA, and ( S, S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of ( S, S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids.

Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol–1. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predo...

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5′-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,β-unsaturated aldehyde of the 5′-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examine...

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s−1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and “gap-filling” DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the e-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol β having a lysine analogue at position 72. Replacement of the primary n...

Menaquinone (MK, vitamin K) is a lipid-soluble quinone that participates in the bacterial electron transport chain. In mammalian cells, vitamin K functions as an essential vitamin for the activation of several proteins involved in blood clotting and bone metabolism. MqnA is the first enzyme on the futalosine-dependent pathway to menaquinone and catalyzes the aromatization of chorismate by water loss. Here we report biochemical and structural studies of MqnA. These studies suggest that the dehydration reaction proceeds by a variant of the E1cb mechanism in which deprotonation is slower than water loss and that the enol carboxylate of the substrate is serving as the base.

LigJ from the soil bacterium Sphingobium sp. SYK-6 catalyzes the reversible hydration of (3Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) in the degradation of lignin in the protocatechuate 4,5-cleavage pathway. LigJ is a member of the amidohydrolase superfamily and an enzyme in cog2159. The three-dimensional crystal structure of wild-type LigJ was determined in the presence [Protein Data Bank (PDB) entry 6DXQ] and absence of the product CHA (PDB entry 6DWV). The protein folds as a distorted (β/α)8-barrel, and a single zinc ion is bound in the active site at the C-terminal end of the central β-barrel. The product CHA is ligated to the zinc ion in the active site via the displacement of a single water molecule from the coordination shell of the metal center in LigJ. The product-bound structure reveals that the enzyme catalyzes the hydration of KCH with the formation of a chiral center at C4 with S stereochemistry. The E284Q mutant was unable to catalyze the hydration of ...

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

As one of the most abundant elements on earth, sulfur is part of many small molecular metabolites and is key to their biological activities. Over the past few decades, some general strategies have been discovered for the incorporation of sulfur into natural products. In this review, we summarize recent efforts in elucidating the biosynthetic details for two sulfur-containing metabolites, ergothioneine and ovothiol. Their biosyntheses involve an unprecedented trans-sulfur strategy, a combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C–S bond formation reaction and a PLP enzyme-mediated C–S lyase reaction.

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg2+ ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of 36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis. © 2015 American Chemical Society., National Institutes of Health (NIH) (GM56838/GM30301) NIH Structural Biology and Molecular Biophysics Training Grant; Radcliffe Institute for Advanced Study

A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.

For almost 40 years, it has been known that tryptophan metabolites and picolinic acid analogues act as inhibitors of gluconeogenesis. Early studies observed that 3-mercaptopicolinic acid (MPA) was a potent hypoglycemic agent via inhibition of glucose synthesis through the specific inhibition of phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenesis pathway. Despite prior kinetic investigation, the mechanism of the inhibition by MPA is unclear. To clarify the mechanism of inhibition exerted by MPA on PEPCK, we have undertaken structural and kinetic studies. The kinetic data in concert with crystallographic structures of PEPCK in complex with MPA and the substrates for the reaction illustrate that PEPCK is inhibited by the binding of MPA at two discrete binding sites: one acting in a competitive fashion with PEP/OAA (∼10 μM) and the other acting at a previously unidentified allosteric site (Ki ∼ 150 μM). The structural studies suggest that binding of MPA to the allosteric pocket stabilizes an altered conformation of the nucleotide-binding site that in turn reduces the affinity of the enzyme for the nucleotide.

The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.

Enormous amounts of the organic osmolyte dimethylsulfoniopropionate (DMSP) are produced in marine environments where bacterial DMSP lyases cleave it, yielding acrylate and the climate-active gas dimethyl sulfide (DMS). SAR11 bacteria are the most abundant clade of heterotrophic bacteria in the oceans and play a key role in DMSP catabolism. An important environmental factor affecting DMS generation via DMSP lyases is the availability of metal ions because they are essential cofactors for many of these enzymes. Here we examine the structure and activity of DddK in the presence of various metal ions. We have established that DddK containing a double-stranded β-helical motif utilizes various divalent metal ions as cofactors for catalytic activity. However, nickel, an abundant metal ion in marine environments, adopts a distorted octahedral coordination environment and conferred the highest DMSP lyase activity. Crystal structures of cofactor-bound DddK reveal key metal ion binding and catalytic residues and provide the first rationalization for varying activities with different metal ions. The structures of DddK along with site-directed mutagenesis and ultraviolet-visible studies are consistent with Tyr 64 acting as a base to initiate the β-elimination reaction of DMSP. Our biochemical and structural studies provide a detailed understanding of DMS generation by one of the ocean's most prolific bacteria.

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 A resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 A inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S](2+) cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal β domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron-sulfur cluster producing a "tail in mouth" configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the β domain are clustered around the iron-sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-β-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-alpha-phenylalanine into (R)-beta-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic acid, a reaction that can be used for the preparation of beta-amino acids, which occur as frequent constituents of bioactive compounds. Different hypotheses have been formulated to explain the stereochemistry of the PAM-catalyzed reaction, but structural evidence for these hypotheses is lacking. Furthermore, it remains unclear how the PAM MIO group is formed from the three-amino acid (A-S-G) sequence motif. For these reasons, we elucidated PAM three-dimensional (3D) structures with a bound (R)-beta-phenylalanine analogue and with bound trans-cinnamic acid. In addition, 3D structures of the (inactive) Y322A and N231A mutants of PAM were elucidated, which were found to be MIO-less. We conclude that the stereochemistry of the PAM-catalyzed reaction originates from the enzyme's ability to bind trans-cinnamic acid in two different orientations, with either the si,si face or the re,re face directed toward the MIO group, as evidenced by two distinct carboxylate binding modes. The results also suggest that the N231 side chain promotes MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton.

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

Soluble guanylate cyclase (sGC) plays a central role in the cardiovascular system and is a drug target for the treatment of pulmonary hypertension. While the three-dimensional structure of sGC is unknown, studies suggest that binding of the regulatory domain to the catalytic domain maintains sGC in an autoinhibited basal state. The activation signal, binding of NO to heme, is thought to be transmitted via the regulatory and dimerization domains to the cyclase domain and unleashes the full catalytic potential of sGC. Consequently, isolated catalytic domains should show catalytic turnover comparable to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we show unambiguously that human isolated catalytic domains are much less active than basal sGC, while still forming heterodimers. We identified key structural elements regulating the dimer interface and propose a novel role for residues located in an interfacial flap and a hydrogen bond network as key modulators of the orientation of the catalytic subunits. We demonstrate that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. Our data support a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of heterodimerization and activation of sGC and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2-100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.

Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1,4-glycoside bonds between uronate and amino sugar residues. Being different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with the active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel β-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report of the structure-function relationship of P. heparinus Hep III.

A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme's β8-α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ's reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of l-tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-β-methyltryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)8TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs. © 2013 American Chemical Society.

The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.

Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa, algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity and revealed that AlgL operates preferentially on nonacetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The k(cat)/K(m) for the reaction increased linearly with the number of residues in the substrate, from 2.1 × 10(5) M(-1) s(-1) for the substrate containing 16 residues to 7.9 × 10(6) M(-1) s(-1) for the substrate with 280 residues. Over the same substrate size range, k(cat) varied between 10 and 30 s(-1). The variation in k(cat)/K(m) with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by (1)H nuclear magnetic resonance spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.

The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ∼300 million tons of dimethyl sulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ, reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily on the basis of structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP β-elimination reaction catalyzed by DddQ as our as-isolated purple-colored enzyme possesses ∼0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with a tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of the 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III) oxidized and Fe(II) reduced species are active, with similar k

Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5–2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or −2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic sidechain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio-function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267 – 0.505 kcal/mol of catalytic free energy, or roughly 6 – 11% rescue).