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 language | English (US) |
---|---|
Pages (from-to) | 4221-4229 |
Number of pages | 9 |
Journal | ACS Catalysis |
Volume | 7 |
Issue number | 6 |
DOIs | |
State | Published - Jun 2 2017 |
Bibliographical note
Publisher Copyright:© 2017 American Chemical Society.
Keywords
- ancestral enzyme
- enantioselectivity
- esterase
- hydroxynitrile lyase
- molecular dynamics
- α/β-hydrolase fold