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Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate.

  • Author(s): Poust, Sean;
  • Yoon, Isu;
  • Adams, Paul D;
  • Katz, Leonard;
  • Petzold, Christopher J;
  • Keasling, Jay D
  • Editor(s): Vertessy, Beata G
  • et al.
Abstract

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.

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