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
Funding Information:We thank the U.S. National Institutes of Health (No. 1R01GM102205-01), the National Science Foundation (CHM-1152804) and the Fulbright Visiting Student Researcher Program to ZB for financial support, the Minnesota Supercomputing Institute for use of computers and software, and Mark Lunzer and Antony Dean for helpful discussions and data for several AtHNL single substitution variants.
Publisher Copyright:
© 2017 American Chemical Society.
Keywords
- ancestral enzyme
- enantioselectivity
- esterase
- hydroxynitrile lyase
- molecular dynamics
- α/β-hydrolase fold