Identical Active Sites in Hydroxynitrile Lyases Show Opposite Enantioselectivity and Reveal Possible Ancestral Mechanism

Bryan J. Jones, Zsófia Bata, Romas J. Kazlauskas

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Evolutionarily related hydroxynitrile lyases from rubber tree (HbHNL) and from Arabidopsis thaliana (AtHNL) follow different catalytic mechanisms with opposite enantioselectivity toward mandelonitrile. We hypothesized that the HbHNL-like mechanism evolved from an enzyme with an AtHNL-like mechanism. We created ancestor-like composite active sites in each scaffold to elucidate how this transition may have occurred. Surprisingly, a composite active site in HbHNL maintained (S)-selectivity, while the identical set of active site residues in AtHNL maintained (R)-selectivity. Composite active-site mutants that are (S)-selective without the Lys236 and Thr11 that are required for the classical (S)-HNL mechanism suggest a new mechanism. Modeling suggested a possibility for this new mechanism that does not exist in modern enzymes. Thus, the last common ancestor of HbHNL and AtHNL may have used an extinct mechanism, not the AtHNL-like mechanism. Multiple mechanisms are possible with the same catalytic residues and residues outside the active site strongly influence the mechanism and enantioselectivity.

Original languageEnglish (US)
Pages (from-to)4221-4229
Number of pages9
JournalACS Catalysis
Volume7
Issue number6
DOIs
StatePublished - Jun 2 2017

Keywords

  • ancestral enzyme
  • enantioselectivity
  • esterase
  • hydroxynitrile lyase
  • molecular dynamics
  • α/β-hydrolase fold

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