Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase

John F. Rakus, Alexander A. Fedorov, Elena V. Fedorov, Margaret E. Glasner, Brian K. Hubbard, Joseph D. Delli, Patricia C. Babbitt, Steven C. Almo, John A. Gerlt

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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 E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; 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 N-terminal 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 Mg2+ 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-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex. His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.

Original languageEnglish (US)
Pages (from-to)9944-9954
Number of pages11
JournalBiochemistry
Volume47
Issue number38
DOIs
StatePublished - Sep 23 2008

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Hydro-Lyases
Phosphopyruvate Hydratase
Catalytic Domain
Salmonella typhimurium
Escherichia coli
Protons
Sugar Acids
Salmonella
Acids
Substrate Specificity
Dehydration
Carbon
Genes
Crystal structure
Genome
Ligands
Catalysts
Kinetics
Substrates
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., ... Gerlt, J. A. (2008). Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase. Biochemistry, 47(38), 9944-9954. https://doi.org/10.1021/bi800914r

Evolution of enzymatic activities in the enolase superfamily : L-rhamnonate dehydratase. / Rakus, John F.; Fedorov, Alexander A.; Fedorov, Elena V.; Glasner, Margaret E.; Hubbard, Brian K.; Delli, Joseph D.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.

In: Biochemistry, Vol. 47, No. 38, 23.09.2008, p. 9944-9954.

Research output: Contribution to journalArticle

Rakus, JF, Fedorov, AA, Fedorov, EV, Glasner, ME, Hubbard, BK, Delli, JD, Babbitt, PC, Almo, SC & Gerlt, JA 2008, 'Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase', Biochemistry, vol. 47, no. 38, pp. 9944-9954. https://doi.org/10.1021/bi800914r
Rakus JF, Fedorov AA, Fedorov EV, Glasner ME, Hubbard BK, Delli JD et al. Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase. Biochemistry. 2008 Sep 23;47(38):9944-9954. https://doi.org/10.1021/bi800914r
Rakus, John F. ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Glasner, Margaret E. ; Hubbard, Brian K. ; Delli, Joseph D. ; Babbitt, Patricia C. ; Almo, Steven C. ; Gerlt, John A. / Evolution of enzymatic activities in the enolase superfamily : L-rhamnonate dehydratase. In: Biochemistry. 2008 ; Vol. 47, No. 38. pp. 9944-9954.
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