Evolution of Enzymatic Activities in the Enolase Superfamily:  Identification of a “New” General Acid Catalyst in the Active Site of <scp>d</scp>-Galactonate Dehydratase from <i>Escherichia coli</i>

Wieczorek, Stacey J.; Kalivoda, Kathryn A.; Clifton, James R; Ringe, Dagmar; Petsko, Gregory A.; Gerlt, J.A.

doi:10.1021/ja990500w

Cited by 36 publications

(46 citation statements)

References 9 publications

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“…An alignment of the sequence of Ob2843 with those of the nine functionally assigned acid sugar dehydratases in the enolase superfamily [D-glucarate dehydrtatase (GlucD; GlucD subgroup (21)), D-mannonate dehydratase (ManD; ManD subgroup (3)), L-fuconate dehydratase (FucD; MR subgroup (22)), D-tartrate dehydratase (TarD; MR subgroup (23)), L-talarate/galactarate dehydratase (TalrD/GalrD; MR subgroup (8)), D-gluconate dehydratase (GluD; MR subgroup (24)), D-arabinonate (AraD; MR subgroup (25)), D-galactonate dehydratase (GalD; MR subgroup (26)), and L-rhamnonate dehydratase (RhamD; MR subgroup (27))] strongly suggested that the active site architecture and catalytic strategy of Ob2843 would be unique:The active site of Ob2843 was not expected to possess homologues of either residue in the His-Asp dyad located at the ends of the seventh and sixth β-strands of the (β/α) 7 β-barrel domain as observed in the dehydratases in the GlucD and MR subgroups.The active site was not expected to contain an electrophilic Lys at the end of the second β-strand that stabilizes the enolate anion intermediate and often occurs as a Lys-x-Lys motif in the dehydratases in the GlucD and MR subgroups, where the first Lys is the electrophile and the second is an acid/base catalyst. Instead, the alignment suggested that an Arg is located at the end of the second β-strand, with a Tyr located two residues toward the C-terminus from the Arg to form an Arg-x-Tyr motif ( vide infra ); ManD contains an Arg at this position but forms an Arg-Tyr dyad with a distal Tyr in the loop that follows the second β-strand (3).…”

Section: Resultsmentioning

confidence: 99%

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis,

et al. 2009

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The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI: 23100298; IMG locus tag Ob2843; PDB Code 2OQY) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg 2+ ions located 10.4 Å from one another, with one located in the canonical position in the (β/ α) 7 β-barrel domain (although the ligand at the end of the fifth β-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono-and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (k cat = 6.8 s −1 , K M = 620 μM; k cat /K M = 1.1 × 10 4 M −1 s −1 ), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II) The structure of a complex of the catalytically impaired Y90F mutant with Mg 2+ and galactarate allowed identification † This research was supported by NIH P01 GM071790 (to J.A.G., M.P.J., and S.C.A.) and NIH U54 GM074945 (to S.K.B.). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). M.P.J. is a consultant to Schrödinger LLC. The X-ray coordinates and structure factors for wild type GalrD-II from Oceanobacillus iheyensis (SeMet-labeled) liganded with Mg 2+ , wild type GalrD-II (SMet) liganded with Mg 2+ , wild type GalrD-II (SMet) liganded with Mg 2+ and L-malate in two space groups, and the Y90F mutant of GalrD-II (SMet) liganded with Mg 2+ and galactarate have been deposited in the Protein Data Bank (PDB Codes 2OQY, 3FYY, 3ES7, 3ES8, and 3HPF, respectively).*To whom correspondence should be addressed: J.A.G.: Department of Biochemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801. Telephone: (217) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the α-proton and Tyr 90 as the acid that facilitates departure of the β-OH leaving group. The enzyme product is 2-keto-3-deoxy-D-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional L-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example that an integrated sequence/structure-based strategy employing computational approaches is a viable appr...

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Section: Resultsmentioning

confidence: 99%

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis,

et al. 2009

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“…However, the sequence alignment also suggests that in YfaW only the electrophilic Lys residue is located at the end of the second β-strand, as was previously observed in the active site of GalD. In GalD, a His at the end of the third β-strand was identified as the acid catalyst that enables the anti-elimination reaction (8); a homologue of this His residue is absent in YfaW. Thus, the reaction catalyzed by YfaW contains a novel active site motif that enables a “new” function in the MR subgroup.…”

Section: Resultsmentioning

confidence: 69%

Evolution of Enzymatic Activities in the Enolase Superfamily: l-Rhamnonate Dehydratase

Rakus

Fedorov

et al. 2008

Biochemistry

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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 NaBH 4 ). Like other members of the enolase superfamily, RhamD contains an Nterminal α+ β capping domain and a C-terminal (β/α) 7 β-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- † This research was supported by GM-52594, P01 GM-71790, U54 GM-74945, and a Ruth L. Kirschstein National Research Service Award 5 T32 GM070421 from the National Institutes of Health. The X-ray coordinates and structure factors for RhamD from Salmonella typhimurium LT2 liganded with Mg 2+ , RhamD from S. typhimurium LT2 liganded with Mg 2 and 3-deoxy-L-rhamnonate, and RhamD from Escherichia coli in the absence of ligands have been deposited in the Protein Data Bank (PDB accession codes, 3BOX, 3CXO, and 2I5Q, respectively).* To whom correspondence should be addressed: J.A.G.: Department of Biochemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801. Phone: (217) 1-3). The members share a conserved tertiary structure with a twodomain architecture, in which three carboxylate ligands for the Mg 2+ ion as well as the acid/ base catalysts are located at the C-terminal ends of the β-strands in a (β/α) 7 β-barrel [modified (β/α) 8 -or TIM-barrel] domain and the specificity-determining residues are located in an Nterminal α+β capping domain.The pseudosymmetric structure of the barrel domain provides a mechanistically versatile scaffold for the catalytic residues (4). Functional groups are positioned on either face of the enediolate intermediate to allow mechanistic variation that is characterized by an array of stereochemical characteristics. The known reactions involve β-elimination of either hydroxide or ammonia leaving groups, 1,1-proton transfer in racemization and epimerization, and intramolec...

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“…9–11,19–21 The overall reaction mechanism is unambiguous, 10,11 proceeding through an enolate intermediate, as is believed to be the case for all known members of the superfamily. In the dehydration of D-glucarate, an active site histidine (H339) abstracts a proton from C5 to give the enediolate intermediate I1 (Scheme 1).…”

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confidence: 83%

Predicting Enzyme–Substrate Specificity with QM/MM Methods: A Case Study of the Stereospecificity of d-Glucarate Dehydratase

et al. 2013

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The stereo-specificity of D-glucarate dehydratase (GlucD) is explored by QM/MM calculations. Both the substrate binding and the chemical steps of GlucD contribute to substrate specificity. Although the identification of transition states remains computationally intensive, we suggest that QM/MM computations on ground states or intermediates can capture aspects of specificity that cannot be obtained using docking or molecular mechanics methods.

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Evolution of Enzymatic Activities in the Enolase Superfamily: Identification of a “New” General Acid Catalyst in the Active Site of d-Galactonate Dehydratase from Escherichia coli

Cited by 36 publications

References 9 publications

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis,

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis,

Evolution of Enzymatic Activities in the Enolase Superfamily: l-Rhamnonate Dehydratase

Predicting Enzyme–Substrate Specificity with QM/MM Methods: A Case Study of the Stereospecificity of d-Glucarate Dehydratase

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