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

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

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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 (b116730; 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 β-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth β-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 β-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 × 104 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 α-proton to generate a Mg2+-stabilized enediolate intermediate, and the vinylogous β-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.

Original languageEnglish (US)
Pages (from-to)14598-14608
Number of pages11
JournalBiochemistry
Volume45
Issue number49
DOIs
StatePublished - Dec 12 2006

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mandelate racemase
Bradyrhizobium
Phosphopyruvate Hydratase
Protons
Oxaloacetic Acid
Acids
Catalytic Domain
Sugar Acids
Kinetics
Proton transfer
Sequence Alignment
Functional groups
Nuclear magnetic resonance spectroscopy
Hydrogen
Magnetic Resonance Spectroscopy
Genes
Observation
Genome
Ligands
Decomposition

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evolution of enzymatic activities in the enolase superfamily : D-tartrate dehydratase from Bradyrhizobium japonicum. / Yew, Wen Shan; Fedorov, Alexander A.; Fedorov, Elena V.; Wood, Bryant McKay; Almo, Steven C.; Gerlt, John A.

In: Biochemistry, Vol. 45, No. 49, 12.12.2006, p. 14598-14608.

Research output: Contribution to journalArticle

Yew, Wen Shan ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Wood, Bryant McKay ; Almo, Steven C. ; Gerlt, John A. / Evolution of enzymatic activities in the enolase superfamily : D-tartrate dehydratase from Bradyrhizobium japonicum. In: Biochemistry. 2006 ; Vol. 45, No. 49. pp. 14598-14608.
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AU - Almo, Steven C.

AU - Gerlt, John A.

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