Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2

Shan Yew Wen, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo, John A. Gerlt

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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 β-strand in the (β/α) 7β-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth β-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth β-strands, and an electrophilic Glu 348 at the end of the eighth β-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 × 103 M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 × 104 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 α-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.

Original languageEnglish (US)
Pages (from-to)9564-9577
Number of pages14
JournalBiochemistry
Volume46
Issue number33
DOIs
StatePublished - Aug 21 2007

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Hydro-Lyases
Salmonella
Phosphopyruvate Hydratase
Salmonella typhimurium
Dehydration
Acids
mandelate racemase
Catalytic Domain
Carbon
talaric acid
Sugar Acids
Gene encoding
Deuterium
Protonation
Metabolites
Protons
Catalyst activity
Screening
Proteins
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evolution of enzymatic activities in the enolase superfamily : L-talarate/galactarate dehydratase from Salmonella typhimurium LT2. / Wen, Shan Yew; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.

In: Biochemistry, Vol. 46, No. 33, 21.08.2007, p. 9564-9577.

Research output: Contribution to journalArticle

Wen, Shan Yew ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Almo, Steven C. ; Gerlt, John A. / Evolution of enzymatic activities in the enolase superfamily : L-talarate/galactarate dehydratase from Salmonella typhimurium LT2. In: Biochemistry. 2007 ; Vol. 46, No. 33. pp. 9564-9577.
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N2 - 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 β-strand in the (β/α) 7β-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth β-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth β-strands, and an electrophilic Glu 348 at the end of the eighth β-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 × 103 M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 × 104 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 α-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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