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John A Gerlt

Publications and source records attributed to John A Gerlt.

31 records · Page 2Linked to original sources
PubMed2004

Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida.

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.

Amino Acid Sequence↗
PubMed2004

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

o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.

Actinomycetales↗
PubMed2003

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli.

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.

Amino Acid Substitution↗
PubMed2003

Chemical and enzymatic synthesis of fluorinated-dehydroalanine-containing peptides.

Michael acceptors have long been recognized as reactive functionalities that may link a biologically active molecule to its cellular target. 1,2-Dehydro amino acids are potential Michael acceptors present in a large number of natural products, but their reactivity is modulated by the deactivating nature of the alpha-amino group engaged in an amide bond. We describe here the preparation of 3-fluoro-1,2-dehydroalanine moieties within peptides that significantly enhance the reactivity of the Michael acceptor. Two different routes were designed to access these compounds, one relying on chemical means to introduce the desired functionality and the second taking advantage of a peptide epimerase. In the chemical approach, the fluoro-Pummerer reaction of cysteine derivatives afforded 3-fluorocysteine residues that were oxidized to the corresponding sulfoxides, followed by thermolytic elimination to provide the desired 3-fluorodehydroalanines. The mechanism of the fluoro-Pummerer reaction was investigated and several possible pathways were ruled out. The enzymatic approach utilized the dipeptide epimerase YcjG from Escherichia coli. Difluorinated alanine was incorporated at the C terminus of a dipeptide by chemical means. The resulting peptide proved to be a substrate for YcjG, which catalyzed fluoride elimination to provide the 3-fluorodehydroalanine-containing peptide. Mechanistic investigations showed that fluoride elimination occurred faster than epimerization and at a rate close to that of epimerization of Ala-Ala.

Alanine↗
PubMed2003

Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates. KGPDC catalyzes the Mg(2+) ion-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to yield L-xylulose 5-phosphate and CO(2); OMPDC catalyzes the metal ion-independent decarboxylation of OMP to UMP and CO(2). Structural studies have shown that KGPDC and OMPDC share several strictly conserved active site residues that are used differently by each enzyme to catalyze their mechanistically distinct reactions. Although the mechanism of the KGPDC-catalyzed reaction has yet to be elucidated, it is thought to proceed via a Mg(2+) ion-stabilized 1,2-enediolate intermediate. Here we report the crystal structures of KGPDC complexed with L-gulonate 6-phosphate, L-threonohydroxamate 4-phosphate, and L-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product, L-xylulose 5-phosphate, at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively. These structures support a mechanism that involves the formation of a cis-1,2-enediolate intermediate. Contrary to expectations, the geometry of the intermediate does not involve bidentate coordination of both enediolate oxygen atoms to the Mg(2+) ion but rather involves only the coordination of the oxygen on C2 to the Mg(2+) ion. The oxygen atom on C1 instead forms hydrogen bonds to both Lys64 and Asp67, two strictly conserved active site residues. Lys64 also interacts with the oxygen on C2 and may serve to stabilize a cis conformation of the 1,2-enediolate. These structures also implicate His136 to be the general acid that protonates the 1,2-enediolate intermediate. This study further demonstrates that multiple unrelated enzyme functions can evolve from a single active site architecture without regard for substrate binding affinity or mechanism.

Carboxy-Lyases↗
PubMed2003

Divergent function in the crotonase superfamily: an anhydride intermediate in the reaction catalyzed by 3-hydroxyisobutyryl-CoA hydrolase.

3-Hydroxyisobutyryl-CoA hydrolase (HICH), a member of the enoyl-CoA (crotonase) superfamily, catalyzes the hydrolysis of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate. Like other members of the superfamily, the sequence of HICH contains conserved sequences for an oxyanion hole that stabilizes anionic intermediates. In contrast to most members of the superfamily, the reaction catalyzed by HICH does not proceed via formation of a thioester enolate anion; instead, evidence based on substrate deuterium isotope effects, the reactivity of substrate analogues that cannot form thioester enolate anions, single-turnover experiments in H218O, and the kinetic phenotypes of site-directed mutants provide evidence for a mechanism involving the formation of an anhydride intermediate involving Glu143 in the active site. In the reactions catalyzed by many members of the superfamily, homologues of Glu143 abstract the alpha proton of the thioester substrate to generate the thioester enolate anion intermediate. Presumably, one or more of the anionic tetrahedral intermediates on the HICH reaction coordinate are stabilized by the oxyanion hole. Thus, we conclude that the conserved oxyanion hole in this superfamily can be used to stabilize a variety of anionic intermediates.

Anhydrides↗
PubMed2003

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily.

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.

Amino Acid Sequence↗
PubMed2003

How to find "missing" genes.

Assigning function to "new" proteins is frequently the rate-determining step for deciphering metabolic pathways and regulatory networks. Osterman and Begley break down this barrier by demonstrating that comparative analyses of microbial genomes is a powerful strategy for identifying pathway components.

Bacteria↗
PubMed2003

Evolution of function in (beta/alpha)8-barrel enzymes.

The (beta/alpha)(8)-barrel is the most common fold in structurally characterized enzymes. Whether the functionally diverse enzymes that share this fold are the products of either divergent or convergent evolution (or both) is an unresolved question that will probably be answered as the sequence databases continue to expand. Recent work has examined natural, designed, and directed evolution of function in several superfamilies of (beta/alpha)(8)-barrel containing enzymes.

Animals↗
PubMed2003

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.

Aldehyde Oxidoreductases↗
PubMed2002

Homologous (beta/alpha)8-barrel enzymes that catalyze unrelated reactions: orotidine 5'-monophosphate decarboxylase and 3-keto-L-gulonate 6-phosphate decarboxylase.

The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) encoded by the ulaD gene in the Escherichia coli genome [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306] and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous (derived from a common ancestor) but catalyze different reactions. The metal-independent decarboxylation reaction catalyzed by OMPDC avoids the formation of a vinyl anion intermediate; the Mg2+-dependent decarboxylation reaction catalyzed by KGPDC involves the formation of an enediolate anion intermediate. Based on the available structures of OMPDC, a sequence alignment allows the predictions that (1) KGPDC is a dimer of (beta/alpha)8-barrels, with the active sites located at the dimer interface; (2) KGPDC and OMPDC share an aspartate residue at the end of the first beta-strand and an Asp-x-Lys-x-x-Asp motif at the end of the third beta-strand with OMPDC; but (3) KGPDC has a Glu instead of a Lys at the end of the second beta-strand. The structure of KGPDC has been determined in the presence of Mg2+ and the substrate analogue L-gulonate 6-phosphate and confirms these predictions. The carboxylate functional groups at the ends of the first, second, and third beta-strands in KGPDC are ligands of the Mg2+; in OMPDC, the homologues of these residues participate in a hydrogen-bonded network that facilitates the decarboxylation reaction. The 3-OH group of the substrate analogue is coordinated to the Mg2+, supporting the hypothesis that the mechanism of the decarboxylation catalyzed by KGPDC involves stabilization of an enediolate anion intermediate. These structural studies establish the existence of the OMPDC "suprafamily," in which members catalyze reactions that occur in different metabolic pathways and share no mechanistic relationship. The existence of this suprafamily demonstrates that divergent evolution can be opportunistic, conscripting active site features of a progenitor to catalyze unrelated functions. Accordingly, sequence or structure homology alone cannot be used to infer the functions of new proteins discovered in genome projects.

Amino Acid Sequence↗
PubMed2002

Utilization of L-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons.

Escherichia coli K-12 can ferment L-ascorbate. The operon encoding catabolic enzymes in the utilization of L-ascorbate (ula) has been identified; this operon of previously unknown function had been designated the yif-sga operon. Three enzymes in the pathway that produce D-xylulose 5-phosphate have been functionally characterized: 3-keto-L-gulonate 6-phosphate decarboxylase (UlaD), L-xylulose 5-phosphate 3-epimerase (UlaE), and L-ribulose 5-phosphate 4-epimerase (UlaF). Several products of the yia-sgb operon were also functionally characterized, although the substrate and physiological function of the operon remain unknown: 2,3-diketo-L-gulonate reductase (YiaK), 3-keto-L-gulonate kinase (LyxK), 3-keto-L-gulonate 6-phosphate decarboxylase (SgbH), and L-ribulose 5-phosphate 4-epimerase (SgbE).

Ascorbic Acid↗