Biomedical subjects

John A Gerlt

Publications and source records attributed to John A Gerlt.

At least 19 recordsLinked to original sources

PubMed2007

Mechanisms of protein evolution and their application to protein engineering.

Protein engineering holds great promise for the development of new biosensors, diagnostics, therapeutics, and agents for bioremediation. Despite some remarkable successes in experimental and computational protein design, engineered proteins rarely achieve the efficiency or specificity of natural enzymes. Current protein design methods utilize evolutionary concepts, including mutation, recombination, and selection, but the inability to fully recapitulate the success of natural evolution suggests that some evolutionary principles have not been fully exploited. One aspect of protein engineering that has received little attention is how to select the most promising proteins to serve as templates, or scaffolds, for engineering. Two evolutionary concepts that could provide a rational basis for template selection are the conservation of catalytic mechanisms and functional promiscuity. Knowledge of the catalytic motifs responsible for conserved aspects of catalysis in mechanistically diverse superfamilies could be used to identify promising templates for protein engineering. Second, protein evolution often proceeds through promiscuous intermediates, suggesting that templates which are naturally promiscuous for a target reaction could enhance protein engineering strategies. This review explores these ideas and alternative hypotheses concerning protein evolution and engineering. Future research will determine if application of these principles will lead to a protein engineering methodology governed by predictable rules for designing efficient, novel catalysts.

Binding Sites↗

PubMed2006

Evolution of enzymatic activities in the enolase superfamily: L-fuconate dehydratase from Xanthomonas campestris.

Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report we use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI:21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of the third, fourth, and fifth beta-strands in the (beta/alpha)7beta-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second beta-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and beta-strands (His 351 and Asp 324, respectively), and a Glu at the end of the eighth beta-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous beta-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting enol, likely by the conjugate acid of Lys 220, to yield the 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L-galactonate and D-arabinonate in competition with dehydration. The functional promiscuity discovered for FucD highlights possible structural mechanisms for evolution of function in the enolase superfamily.

Amino Acid Substitution↗

PubMed2006

Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum.

We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second beta-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth beta-strands, respectively, as well as ligands for an essential Mg2+, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth beta-strands, respectively. We screened a library of 46 acid sugars and discovered that only d-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (kcat = 7.3 s(-1); kcat/KM = 8.5 x 10(4) M(-1) s(-1)) are consistent with assignment of the d-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the alpha-proton to generate a Mg2+-stabilized enediolate intermediate, and the vinylogous beta-elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by 1H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a "simple" extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg2+-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.

Bacterial Proteins↗

PubMed2006

Evolution of enzyme superfamilies.

Enzyme evolution is often constrained by aspects of catalysis. Sets of homologous proteins that catalyze different overall reactions but share an aspect of catalysis, such as a common partial reaction, are called mechanistically diverse superfamilies. The common mechanistic steps and structural characteristics of several of these superfamilies, including the enolase, Nudix, amidohydrolase, and haloacid dehalogenase superfamilies have been characterized. In addition, studies of mechanistically diverse superfamilies are helping to elucidate mechanisms of functional diversification, such as catalytic promiscuity. Understanding how enzyme superfamilies evolve is vital for accurate genome annotation, predicting protein functions, and protein engineering.

Binding Sites↗

PubMed2006

Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily.

Understanding how proteins evolve to provide both exquisite specificity and proficient activity is a fundamental problem in biology that has implications for protein function prediction and protein engineering. To study this problem, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase (OSBS/NAAAR) family, part of the mechanistically diverse enolase superfamily. Although all characterized members of the family catalyze the OSBS reaction, this family is extraordinarily divergent, with some members sharing <15% identity. In addition, a member of this family, Amycolatopsis OSBS/NAAAR, is promiscuous, catalyzing both dehydration and racemization. Although the OSBS/NAAAR family appears to have a single evolutionary origin, no sequence or structural motifs unique to this family could be identified; all residues conserved in the family are also found in enolase superfamily members that have different functions. Based on their species distribution, several uncharacterized proteins similar to Amycolatopsis OSBS/NAAAR appear to have been transmitted by lateral gene transfer. Like Amycolatopsis OSBS/NAAAR, these might have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. In addition to functional differences, the OSBS/NAAAR family exhibits surprising structural variations, including large differences in orientation between the two domains. These results offer several insights into protein evolution. First, orthologous proteins can exhibit significant structural variation, and specificity can be maintained with little conservation of ligand-contacting residues. Second, the discovery of a set of proteins similar to Amycolatopsis OSBS/NAAAR supports the hypothesis that new protein functions evolve through promiscuous intermediates. Finally, a combination of evolutionary, structural, and sequence analyses identified characteristics that might prime proteins, such as Amycolatopsis OSBS/NAAAR, for the evolution of new activities.

Actinobacteria↗

PubMed2006

Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids.

Members of the mechanistically diverse enolase superfamily catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion to generate an enolate anion intermediate that is stabilized by coordination to a Mg2+ ion. The catalytic groups, ligands for an essential Mg2+ and acid/base catalysts, are located in the (beta/alpha)8-barrel domain of the bidomain proteins. The assigned physiological functions in the muconate lactonizing enzyme (MLE) subgroup (Lys acid/base catalysts at the ends of the second and sixth beta-strands in the barrel domain) are cycloisomerization (MLE), dehydration (o-succinylbenzoate synthase; OSBS), and epimerization (L-Ala-D/L-Glu epimerase). We previously studied a putatively promiscuous member of the MLE subgroup with uncertain physiological function from Amycolatopsis that was discovered based on its ability to catalyze the racemization of N-acylamino acids (N-acylamino acid racemase; NAAAR) but also catalyzes the OSBS reaction [OSBS/NAAAR; Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. In this manuscript, we report functional characterization of a homologue of this protein encoded by the genome of Geobacillus kaustophilus as well as two other proteins that are encoded by the same operon, a divergent member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes whose members catalyze the transfer an acyl group from an acyl-CoA donor to an amine acceptor, and a member of the M20 peptidase/carboxypeptidase G2 family. We determined that the member of the GNAT superfamily is succinyl-CoA:D-amino acid N-succinyltransferase, the member of the enolase superfamily is N-succinylamino acid racemase (NSAR), and the member of the M20 peptidase/carboxypeptidase G2 family is N-succinyl-L-amino acid hydrolase. We conclude that (1) these enzymes constitute a novel, irreversible pathway for the conversion of D- to L-amino acids and (2) the NSAR reaction is a new physiological function in the MLE subgroup. The NSAR is also functionally promiscuous and catalyzes an efficient OSBS reaction; intriguingly, the operon for menaquinone biosynthesis in G. kaustophilus does not encode an OSBS, raising the possibility that the NSAR is a bifunctional enzyme rather than an accidentally promiscuous enzyme.

Acyl Coenzyme A↗

PubMed2006

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily.

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.

Amino Acid Motifs↗

PubMed2006

A gold standard set of mechanistically diverse enzyme superfamilies.

Superfamily and family analyses provide an effective tool for the functional classification of proteins, but must be automated for use on large datasets. We describe a 'gold standard' set of enzyme superfamilies, clustered according to specific sequence, structure, and functional criteria, for use in the validation of family and superfamily clustering methods. The gold standard set represents four fold classes and differing clustering difficulties, and includes five superfamilies, 91 families, 4,887 sequences and 282 structures.

Conserved Sequence↗

PubMed2005

Evolutionary potential of (beta/alpha)8-barrels: in vitro enhancement of a "new" reaction in the enolase superfamily.

The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the "new" reaction that would provide a selective advantage and (2) an accompanying loss of the "old" reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. The AEE progenitor did not catalyze the OSBS reaction, but the D297G mutant catalyzed a low level of the OSBS reaction (k(cat), 0.013 s(-)(1); K(m), 1.8 mM; k(cat)/K(m), 7.4 M(-)(1) s(-)(1)) that was sufficient to permit anaerobic growth by an OSBS-deficient strain of E. coli; the level of the progenitor's natural AEE reaction was significantly diminished. Using random mutagenesis and an anaerobic metabolic selection, we now have identified the I19F substitution as an additional mutation that enhances both growth of the OSBS-deficient strain and the kinetic constants for the OSBS reaction (k(cat), 0.031 s(-)(1); K(m), 0.34 mM; k(cat)/K(m), 90 M(-)(1) s(-)(1)). Several other substitutions for Ile 19 also enhanced the level of the OSBS reaction. All of the substitutions substantially decreased the level of the AEE reaction from that possessed by the D297G progenitor. The changes in the kinetic constants for both the OSBS and AEE reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSBS reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support our hypothesis that evolution of "new" functions in the enolase superfamily can occur simply by changes in specificity-determining residues.

Amino Acid Isomerases↗

PubMed2005

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: structural basis for catalytic promiscuity in wild-type and designed mutants of 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and D-arabino-hex-3-ulose 6-phosphate synthase (HPS), members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, catalyze reactions that involve the formation of Mg(2+)-ion stabilized 1,2-enediolate intermediates. The active sites of KGPDC and HPS share several conserved residues, including the presumed ligands for the Mg(2+) and a catalytic histidine residue that has been implicated in protonation of the intermediate in the KGPDC-catalyzed reaction. As reported in the previous manuscript, both enzymes are naturally promiscuous, with KGPDC from Escherichia coli catalyzing a low level of the HPS reaction and the HPS from Methylomonas aminofaciens catalyzing a significant level of the KGPDC reaction. Interestingly, the promiscuous HPS reaction catalyzed by KGPDC can be significantly enhanced by replacing no more than four active site residues from KGPDC reaction with residues from HPS. In this manuscript, we report structural studies of wild-type and mutant KDGPC's that provide a structural explanation for both the natural promiscuity for the HPS reaction and the enhanced HPS activity and diminished KGPDC activity catalyzed by active site mutants.

Alanine↗

PubMed2005

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.

Alanine↗

PubMed2005

Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity.

The members of the mechanistically diverse enolase superfamily catalyze different overall reactions. Each shares a partial reaction in which an active site base abstracts the alpha-proton of the carboxylate substrate to generate an enolate anion intermediate that is stabilized by coordination to the essential Mg(2+) ion; the intermediates are then directed to different products in the different active sites. In this minireview, our current understanding of structure/function relationships in the divergent members of the superfamily is reviewed, and the use of this knowledge for our future studies is proposed.

Amino Acid Sequence↗

PubMed2004

Structure of D-ribulose 5-phosphate 3-epimerase from Synechocystis to 1.6 A resolution.

The crystal structure of D-ribulose 5-phosphate 3-epimerase (RPE) from the cyanobacterium Synechocystis was determined by X-ray crystallography to 1.6 A resolution. The enzyme, which catalyzes the epimerization of D-ribulose 5-phosphate and D-xylulose 5-phosphate, assembles as a hexamer of (beta/alpha)(8)-barrels in the crystallographic asymmetric unit. The active site is highly similar to those of two previously reported RPEs and provides further evidence for essential catalytic roles for several active-site residues.

Binding Sites↗

PubMed2004

Evolution of enzymatic activities in the enolase superfamily: structure of a substrate-liganded complex of the L-Ala-D/L-Glu epimerase from Bacillus subtilis.

The members of the mechanistically diverse enolase superfamily share a bidomain structure formed from a (beta/alpha)7beta-barrel domain [a modified (beta/alpha)8- or TIM-barrel] and a capping domain formed from N- and C-terminal segments of the polypeptide. The active sites are located at the interface between the C-terminal ends of the beta-strands in the barrel domain and two flexible loops in the capping domain. Within this structure, the acid/base chemistry responsible for formation and stabilization of an enediolate intermediate derived from a carboxylate anion substrate and the processing of it to product is "hard-wired" by functional groups at the C-terminal ends of the beta-strands in the barrel domain; the identity of the substrate is determined in part by the identities of residues located at the end of the eighth beta-strand in the barrel domain and two mobile loops in the capping domain. On the basis of the identities of the acid/base functional groups at the ends of the beta-strands, the currently available structure-function relationships derived from functionally characterized members are often sufficient for "deciphering" the identity of the chemical reaction catalyzed by sequence-divergent members discovered in genome projects. However, insufficient structural information for liganded complexes for specifying the identity of the substrate is available. In this paper, the structure of the complex of L-Ala-L-Glu with the L-Ala-D/L-Glu epimerase from Bacillus subtilis is reported. As expected for the 1,1-proton transfer reaction catalyzed by this enzyme, the alpha-carbon of the substrate is located between Lys 162 and Lys 268 at the ends of the second and sixth beta-strands in the barrel domain. The alpha-ammonium group of the l-Ala moiety is hydrogen bonded to both Asp 321 and Asp 323 at the end of the eighth beta-strand, revealing a novel strategy for substrate recognition in the superfamily. The delta-carboxylate group of the Glu moiety is hydrogen bonded to Arg 24 in one of the flexible loops in the capping domain, thereby providing a structural explanation for the restricted substrate specificity of this epimerase [Schmidt, D. M., Hubbard, B. K., and Gerlt, J. A. (2001) Biochemistry 40, 15707-15715]. These studies provide important new information about the structural bases for substrate specificity in the enolase superfamily.

Amino Acid Isomerases↗

PubMed2004

Evolution of function in the crotonase superfamily: the stereochemical course of the reaction catalyzed by 2-ketocyclohexanecarboxyl-CoA hydrolase.

Members of the mechanistically diverse enoyl-CoA hydratase (crotonase) superfamily catalyze reactions that involve stabilization of an enolate anion derived from an acyl thioester of coenzyme A. 2-Ketocyclohexanecarboxyl-CoA hydrolase (BadI), found in a pathway for anaerobic degradation of benzoate by Rhodopseudomonas palustris, is a member of the crotonase superfamily that catalyzes a reverse Dieckmann reaction in which the substrate is hydrolyzed to pimelyl-CoA. The substrate is the configurationally labile 2S-ketocyclohexanecarboxyl-CoA, and in 2H2O solvent hydrogen is incorporated into the 2-proS position of the pimelyl-CoA product. Therefore, the stereochemical course of the BadI-catalyzed reaction is inversion. This information is important for understanding the roles of active-site functional groups in the active site of BadI as well as in the active sites of the homologous 1,4-dihydroxynaphthoyl-CoA synthases that catalyze a forward Dieckmann reaction.

Acyl Coenzyme A↗

PubMed2004

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: crystallographic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC), a member of the orotidine monophosphate decarboxylase (OMPDC) suprafamily, catalyzes the Mg(2+)-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose 5-phosphate. Structural and biochemical evidence suggests that the KGPDC reaction proceeds via a Mg(2+)-stabilized 1,2-cis-enediolate intermediate. Protonation of the enediolate intermediate occurs in a nonstereospecific manner to form L-xylulose 5-phosphate. Although the exact mechanism of proton delivery is not known, Glu112, His136, and Arg139 have been implicated in this process [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437]. Surprisingly, single amino acid substitutions of these positions do not substantially reduce catalytic activity but rather alter the stereochemical course of the reaction. Here, we report the X-ray crystal structures of four mutants, K64A, H136A, E112Q, and E112Q/H136A, each determined in the presence of L-threonohydroxamate 4-phosphate, an analogue of the enediolate intermediate, to 1.7, 1.9, 1.8, and 1.9 A resolution, respectively. These structures reveal that substitutions of Lys64, Glu112, and His136 cause changes in the positions of the intermediate analogue and two active site water molecules that were previously identified as possible proton donors. These changes correlate with the observed alterations in the reaction stereochemistry for these mutants, thereby supporting a reaction mechanism in which water molecules competitively shuttle protons from the side chains of His136 and Arg139 to alternate faces of the cis-enediolate intermediate. These studies further underscore the wide variation in the reaction mechanisms in the OMPDC suprafamily.

Binding Sites↗

PubMed2004

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: mechanistic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous enzymes that share the (beta/alpha)(8)-fold but catalyze mechanistically distinct reactions [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869]. KGPDC catalyzes the Mg(2+)-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate, an intermediate in the catabolic pathway of L-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306]. OMPDC catalyzes a metal ion-independent reaction that likely proceeds without a vinyl anion intermediate [Appleby, T. C., Kinsland, C., Begley, T., and Ealick, S. E. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2005-2010], although the mechanistic details are uncertain. An active site Lys located at the end of the third beta-strand in OMPDC has been proposed to be the general acid that delivers a solvent-derived proton to the UMP product; the active site of KGPDC contains a homologous Lys residue (Lys64). Herein, we report investigations of the KGPDC-catalyzed reaction that are consistent with a mechanism involving a Mg(2+)-stabilized cis-enediolate intermediate [Wise, E. L., Yew, W. S., Gerlt, J. A., and Rayment, I. (2003) Biochemistry 42, 12133-12142] and implicate waters proximal to His136 and Arg139, both located at the end of the sixth beta-strand, as the general acids that deliver a solvent-derived proton to the intermediate to form the L-xylulose 5-phosphate product. On the basis of our mechanistic investigations, Lys64 stabilizes the cis-enediolate intermediate by forming hydrogen bonds to both O1 and O2 of the intermediate. Thus, although the active sites of OMPDC and KGPDC contain a conserved Lys at the end of the third beta-strand, their roles in catalysis are not conserved. Furthermore, a conserved Asp at the end of the third beta-strand in OMPDC participates in a hydrogen-bonded network that positions the acidic Lys residue; in the active site of KGPDC, the homologous Asp67 participates in stabilization of the enediolate intermediate and enforces a cis geometry. We conclude that the conserved active site residues perform different functions in the OMPDC- and KGPDC-catalyzed reactions, so the mechanisms of their reactions are completely distinct. This study further highlights the opportunistic nature of divergent evolution in conscripting the active site of a progenitor to catalyze a mechanistically distinct reaction.

Amino Acid Sequence↗

PubMed2004

Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

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