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J A Gerlt - One of the best experts on this subject based on the ideXlab platform.
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Investigating the Physiological Roles of Low-Efficiency D‑Mannonate and D‑Gluconate Dehydratases in the Enolase Superfamily: Pathways for the Catabolism of L‑Gulonate and L‑Idonate
2016Co-Authors: Daniel J. Wichelecki, Steven C Almo, Nawar Al-obaidi, Jean Alyxa Ferolin Vendiola, Amy M. Jones, J A GerltAbstract:ABSTRACT: The sequence/function space in the D-mannonate dehydra-tase subgroup (ManD) of the Enolase Superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722−2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10 3−104 M−1 s−1) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10−102 M−1 s−1) for dehydration of D-mannonate and/or D-gluconate, and no activity. Character-ization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087−4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serova
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2001. Evolution of enzymatic activities in the Enolase Superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. Biochemistry 40:15707–15715
2015Co-Authors: Vadim A. Klenchin, J A Gerlt, Erika Taylor A Ringia, Ivan RaymentAbstract:ABSTRACT: 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′-carboxy-phenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg2+âOSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the Superfamily: the essential Mg2+ is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth â-strands of the (â/R)7â-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second â-strand and the Lys 235 at the end of the sixth â-strand [Thompson, T. B.
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A unique cis -3-hydroxy- l -proline dehydratase in the Enolase Superfamily
Journal of the American Chemical Society, 2015Co-Authors: Xinshuai Zhang, Steven C Almo, Matthew P Jacobson, Ritesh Kumar, Matthew W. Vetting, Suwen Zhao, J A GerltAbstract:The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the Enolase Superfamily (UniProt ID A0NXQ8). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase Superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.
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Discovery of a Novel l‑Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1
2015Co-Authors: Salehe Ghasempur, Steven C Almo, B Hillerich, Subramanyam Swaminathan, Subramaniam Eswaramoorthy, Ronald D. Seidel, J A GerltAbstract:The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the Enolase Superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼104 M–1 s–1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on l-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of l-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-l-lyxonate product by 2-keto-3-deoxy-l-lyxonate dehydratase to yield α-ketoglutarate semialdehyde. In the final step, α-ketoglutarate semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg2+ in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth β-strands of the (β/α)7β-barrel domain as well as a conserved KxR motif at the end of second β-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the Enolase Superfamily and a pathway for the catabolism of l-lyxonate
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Investigating the Physiological Roles of Low-Efficiency d‑Mannonate and d‑Gluconate Dehydratases in the Enolase Superfamily: Pathways for the Catabolism of l‑Gulonate and l‑Idonate
2015Co-Authors: Daniel J. Wichelecki, Steven C Almo, Nawar Al-obaidi, Jean Alyxa Ferolin Vendiola, Amy M. Jones, J A GerltAbstract:The sequence/function space in the d-mannonate dehydratase subgroup (ManD) of the Enolase Superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722–2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 103–104 M–1 s–1) for dehydration of d-mannonate, low-efficiency (kcat/KM = 10–102 M–1 s–1) for dehydration of d-mannonate and/or d-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the d-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087–4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for l-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for l-gulonate catabolism in Escherichia coli CFT073 and l-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential
Steven C Almo - One of the best experts on this subject based on the ideXlab platform.
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Investigating the Physiological Roles of Low-Efficiency D‑Mannonate and D‑Gluconate Dehydratases in the Enolase Superfamily: Pathways for the Catabolism of L‑Gulonate and L‑Idonate
2016Co-Authors: Daniel J. Wichelecki, Steven C Almo, Nawar Al-obaidi, Jean Alyxa Ferolin Vendiola, Amy M. Jones, J A GerltAbstract:ABSTRACT: The sequence/function space in the D-mannonate dehydra-tase subgroup (ManD) of the Enolase Superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722−2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10 3−104 M−1 s−1) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10−102 M−1 s−1) for dehydration of D-mannonate and/or D-gluconate, and no activity. Character-ization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087−4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serova
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A unique cis -3-hydroxy- l -proline dehydratase in the Enolase Superfamily
Journal of the American Chemical Society, 2015Co-Authors: Xinshuai Zhang, Steven C Almo, Matthew P Jacobson, Ritesh Kumar, Matthew W. Vetting, Suwen Zhao, J A GerltAbstract:The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the Enolase Superfamily (UniProt ID A0NXQ8). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase Superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.
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Discovery of a Novel l‑Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1
2015Co-Authors: Salehe Ghasempur, Steven C Almo, B Hillerich, Subramanyam Swaminathan, Subramaniam Eswaramoorthy, Ronald D. Seidel, J A GerltAbstract:The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the Enolase Superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼104 M–1 s–1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on l-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of l-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-l-lyxonate product by 2-keto-3-deoxy-l-lyxonate dehydratase to yield α-ketoglutarate semialdehyde. In the final step, α-ketoglutarate semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg2+ in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth β-strands of the (β/α)7β-barrel domain as well as a conserved KxR motif at the end of second β-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the Enolase Superfamily and a pathway for the catabolism of l-lyxonate
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A Unique cis-3-Hydroxy‑l‑proline Dehydratase in the Enolase Superfamily
2015Co-Authors: Xinshuai Zhang, Steven C Almo, Matthew P Jacobson, Ritesh Kumar, Matthew W. Vetting, Suwen Zhao, J A GerltAbstract:The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the Enolase Superfamily (UniProt ID A0NXQ8). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase Superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources
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Identification of the in Vivo Function of the High-Efficiency d‑Mannonate Dehydratase in Caulobacter crescentus NA1000 from the Enolase Superfamily
2015Co-Authors: Daniel J. Wichelecki, Steven C Almo, Dylan C. Graff, Nawar Al-obaidi, J A GerltAbstract:The d-mannonate dehydratase (ManD) subgroup of the Enolase Superfamily contains members with varying catalytic activities (high-efficiency, low-efficiency, or no activity) that dehydrate d-mannonate and/or d-gluconate to 2-keto-3-deoxy-d-gluconate [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722–2731]. Despite extensive in vitro characterization, the in vivo physiological role of a ManD has yet to be established. In this study, we report the in vivo functional characterization of a high-efficiency ManD from Caulobacter crescentus NA1000 (UniProt entry B8GZZ7) by in vivo discovery of its essential role in d-glucuronate metabolism. This in vivo functional annotation may be extended to ∼50 additional proteins
Ja Gerlt - One of the best experts on this subject based on the ideXlab platform.
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Evolution of enzymatic activities in the Enolase Superfamily: Galactarate dehydratase III from agrobacterium tumefaciens C58
eScholarship University of California, 2014Co-Authors: Jacobson Matthew, Steven C Almo, Fp Groninger-poe, Jt Bouvier, Mw Vetting, Kalyanaraman C, Kumar R, Mp Jacobson, Ja GerltAbstract:The genome of Agrobacterium tumefaciens C58 encodes 12 members of the Enolase Superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars fo
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evolutionary potential of β α 8 barrels stepwise evolution of a new reaction in the Enolase Superfamily
Biochemistry, 2007Co-Authors: Jacob E. Vick, Ja GerltAbstract:The molecular details of the processes involved in divergent evolution of "new" enzymatic functions are ill-defined. Likely starting points are either a progenitor promiscuous for the new reaction or a progenitor capable of catalyzing the new reaction following a single substitution that results from a single base change. However, the molecular (sequence) pathway by which the selective advantage provided by this protein can be improved and ultimately optimized is unclear. In the mechanistically diverse Enolase Superfamily, we discovered that a monofunctional progenitor could acquire the ability to catalyze a "new" reaction by a single base change: the D297G mutant of the monofunctional L-Ala-D/L-Glu epimerase (AEE) from Escherichia coli catalyzed a low level of the o-succinylbenzoate synthase (OSBS) reaction as well as a reduced level of the AEE 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]. We then discovered that the selective advantage and OSBS activity of the D297G mutant are both enhanced by the I19F substitution [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722-11729]. Both the D297G and I19F substitutions are positioned to alter the substrate specificity so that the substrate for the OSBS reaction is more productively positioned vis a vis the active site catalytic groups. We now report that both the selective advantage and OSBS activity of the D297G/ I19F double mutant are enhanced by the R24C (one base change from the wild type Arg codon), R24W (two base changes from the wild type Arg codon and one base change from the R24C codon), and L277W (one base change from the wild type Leu codon) substitutions. The effects of the R24C and L277W mutants are "additive" in the D297G/I19F/R24C/L277W mutant. The greatest selective advantage and OSBS activity are associated with the D297G/I19F/R24W mutant. These "new" substitutions that enhance both the selective advantage and kinetic constants are positioned in the active site where they can alter the specificity, highlighting that the evolution of the "new" OSBS function can be accomplished by changes in substrate specificity.
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evolution of enzymatic activities in the Enolase Superfamily l talarate galactarate dehydratase from salmonella typhimurium lt2
Biochemistry, 2007Co-Authors: Wen Shan Yew, Alexander A Fedorov, E V Fedorov, Steven C Almo, Ja GerltAbstract:We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse Enolase Superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.
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evolution of enzymatic activities in the Enolase Superfamily l fuconate dehydratase from xanthomonas campestris
Biochemistry, 2006Co-Authors: A A Fedorov, E V Fedorov, Steven C Almo, John F. Rakus, Richard W. Pierce, Ja GerltAbstract: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 β-strands in the (β/α)7β-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second β-strand (Lys 2...
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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
Biochemistry, 2006Co-Authors: Ayano Sakai, Dao Feng Xiang, Ling Song, Wen Shan Yew, Frank M. Raushel, Ja GerltAbstract: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.
Ivan Rayment - One of the best experts on this subject based on the ideXlab platform.
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2001. Evolution of enzymatic activities in the Enolase Superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. Biochemistry 40:15707–15715
2015Co-Authors: Vadim A. Klenchin, J A Gerlt, Erika Taylor A Ringia, Ivan RaymentAbstract:ABSTRACT: 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′-carboxy-phenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg2+âOSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the Superfamily: the essential Mg2+ is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth â-strands of the (â/R)7â-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second â-strand and the Lys 235 at the end of the sixth â-strand [Thompson, T. B.
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Divergent evolution in the Enolase Superfamily: The interplay of mechanism and specificity
Archives of biochemistry and biophysics, 2005Co-Authors: J A Gerlt, Patricia C Babbitt, Ivan RaymentAbstract:Abstract 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 α-proton of the carboxylate substrate to generate an enolate anion intermediate that is stabilized by coordination to the essential Mg2+ 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.
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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†,‡
Biochemistry, 2004Co-Authors: Vadim A. Klenchin, J A Gerlt, Dawn M. Z. Schmidt, Ivan RaymentAbstract:The members of the mechanistically diverse Enolase Superfamily share a bidomain structure formed from a (β/α)7β-barrel domain [a modified (β/α)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 β-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 β-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 β-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 β-strands, the currently available structure−fun...
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Evolution of enzymatic activity in the Enolase Superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.
Biochemistry, 2004Co-Authors: James B Thoden, J A Gerlt, James B. Garrett, Erika A Taylor Ringia, Hazel M Holden, Ivan RaymentAbstract: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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evolution of enzymatic activity in the Enolase Superfamily structural studies of the promiscuous o succinylbenzoate synthase from amycolatopsis
Biochemistry, 2004Co-Authors: Erika Taylor A Ringia, James B. Garrett, James B Thoden, Hazel M Holden, Ivan Rayment, Ja GerltAbstract: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.
Patricia C Babbitt - One of the best experts on this subject based on the ideXlab platform.
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Divergent Evolution in Enolase Superfamily: Strategies for Assigning Functions
The Journal of biological chemistry, 2011Co-Authors: J A Gerlt, Patricia C Babbitt, Matthew P Jacobson, Steven C AlmoAbstract:Nature's strategies for evolving catalytic functions can be deciphered from the information contained in the rapidly expanding protein sequence databases. However, the functions of many proteins in the protein sequence and structure databases are either uncertain (too divergent to assign function based on homology) or unknown (no homologs), thereby limiting the utility of the databases. The mechanistically diverse Enolase Superfamily is a paradigm for understanding the structural bases for evolution of enzymatic function. We describe strategies for assigning functions to members of the Enolase Superfamily that should be applicable to other superfamilies.
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discovery of a dipeptide epimerase enzymatic function guided by homology modeling and virtual screening
Structure, 2008Co-Authors: Chakrapani Kalyanaraman, Heidi Imker, Alexander A Fedorov, E V Fedorov, Margaret E Glasner, Patricia C Babbitt, Steven C Almo, J A Gerlt, Matthew P JacobsonAbstract:We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse Enolase Superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function. This prediction was experimentally confirmed, resulting in the assignment of epimerase activity for L-Ala-D/L-Phe, L-Ala-D/L-Tyr, and L-Ala-D/L-His, whereas the enzyme is annotated incorrectly in GenBank as muconate cycloisomerase. Subsequently, crystal structures of the enzyme were determined in complex with three substrates, showing close agreement with the computational models and revealing the structural basis for the observed substrate selectivity.
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Evolution of Enzymatic Activities in the Enolase Superfamily: l-Rhamnonate Dehydratase†‡
Biochemistry, 2008Co-Authors: John F. Rakus, Alexander A Fedorov, Margaret E Glasner, Patricia C Babbitt, Steven C Almo, Elena V. Fedorov, Brian K. Hubbard, Joseph D. Delli, J A GerltAbstract:The L-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse Enolase Superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: L-rhamnonate (6-deoxy-L-mannonate) has the “best” kinetic constants, with L-mannonate, L-lyxonate, and D-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both Escherichia coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg +2 ; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-L-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the Enolase Superfamily, RhamD contains an Nterminal α+ β capping domain and a C-terminal (β/α)7β-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining “20s loop” in the capping domain is extended in length and the “50s loop” is truncated. The ligands for the Mg 2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth β-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth β-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of L
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Evolution of enzymatic activities in the Enolase Superfamily: D-Mannonate dehydratase from Novosphingobium aromaticivorans.
Biochemistry, 2007Co-Authors: John F. Rakus, Alexander A Fedorov, Margaret E Glasner, Patricia C Babbitt, Steven C Almo, Elena V. Fedorov, Jacob E. Vick, J A GerltAbstract:The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse Enolase Superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the Enolase Superfamily, ManD contains two domains, an N-terminal alpha+beta capping domain and a (beta/alpha)7beta-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth beta-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second beta-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth beta-strands, respectively, that are found in many members of the Superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second beta-strand and Arg 147 at the end of the second beta-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third beta-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse Enolase Superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only conserved residues in the Enolase Superfamily, establishing the primary functional importance of the Mg2+-assisted strategy for stabilizing the enolate anion intermediate.
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Prediction and assignment of function for a divergent N-succinyl amino acid racemase
Nature Chemical Biology, 2007Co-Authors: Ling Song, Chakrapani Kalyanaraman, Heidi Imker, Alexander A Fedorov, Margaret E Glasner, Patricia C Babbitt, Steven C Almo, Elena V. Fedorov, Shoshana Brown, Matthew P JacobsonAbstract:The protein databases contain many proteins with unknown function. A computational approach for predicting ligand specificity that requires only the sequence of the unknown protein would be valuable for directing experiment-based assignment of function. We focused on a family of unknown proteins in the mechanistically diverse Enolase Superfamily and used two approaches to assign function: (i) enzymatic assays using libraries of potential substrates, and (ii) in silico docking of the same libraries using a homology model based on the most similar (35% sequence identity) characterized protein. The results matched closely; an experimentally determined structure confirmed the predicted structure of the substrate-liganded complex. We assigned the N -succinyl arginine/lysine racemase function to the family, correcting the annotation ( L -Ala- D/L -Glu epimerase) based on the function of the most similar characterized homolog. These studies establish that ligand docking to a homology model can facilitate functional assignment of unknown proteins by restricting the identities of the possible substrates that must be experimentally tested.