Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

Bijoy J. Desai, Yuki Goto, Alessandro Cembran, Alexander A. Fedorov, Steven C. Almo, Jiali Gao, Hiroaki Suga, John A. Gerlt

Research output: Contribution to journalArticle

Abstract

Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

Original languageEnglish (US)
Pages (from-to)15066-15071
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number42
DOIs
StatePublished - Oct 21 2014

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Keywords

  • Cell-free translation
  • Enzymology
  • Flexible tRNA acylation ribozyme
  • Unnatural protein residue

ASJC Scopus subject areas

  • General

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