Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from staphylococcus aureus

Chao Chen, Jeong Chan Joo, Greg Brown, Ekaterina Stolnikova, Andrei S. Halavaty, Alexei Savchenko, Wayne F. Anderson, Alexander F. Yakunin*

*Corresponding author for this work

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD+ binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

Original languageEnglish (US)
Pages (from-to)3992-4002
Number of pages11
JournalApplied and Environmental Microbiology
Volume80
Issue number13
DOIs
StatePublished - 2014

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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