Abstract
Ketoreductases are a prominent member of the oxidoreductase family with important applications in biotechnology and metabolic engineering, providing a general method for reversible and stereoselective conversion of C═O and C-OH functional groups. As such, developing a deeper understanding of their substrate selectivity would expand our ability to engineer the enzymatic or microbial production of small-molecule targets. Here, we report the crystal structure and biochemical characterization of a mitochondrial ketoreductase AsHadh2 from Ascaris suum with preference for an α-methyl branched substrate, (S)-3-oxo-2-methylbutyryl-CoA (OMB-CoA), compared to its linear analog, 3-oxo-butyryl-CoA (OB-CoA). From docking studies, we found that the α-methyl group appears to be stabilized by a hydrophobic pocket formed by four residues, I155, A156, I199, and M258. Using a combination of saturation mutagenesis at 14 positions surrounding the acyl-CoA substrate and in vivo screening, we identified a set of mutants that alter the α-methyl group selectivity. Mutation of I155 to the smaller A or T increased the OMB-CoA:OB-CoA selectivity by four- to five-fold over wild-type AsHadh2. In contrast, the R213S mutation within the substrate lid lowered α-methyl group selectivity by 2.7-fold. Further characterization shows that for the most part, changes in selectivity are related to tuning the kinetic parameters for the linear OB-CoA substrate and may be related to changes in dynamics in the active site. Taken together, these studies yield insights into the recognition of alkyl substituents and the factors that impact substrate selectivity in enzymatic systems.
Original language | English (US) |
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Pages (from-to) | 8948-8955 |
Number of pages | 8 |
Journal | ACS Catalysis |
Volume | 11 |
Issue number | 14 |
DOIs | |
State | Published - 2021 |
Externally published | Yes |
All Science Journal Classification (ASJC) codes
- Catalysis
- General Chemistry
Keywords
- crystallography
- enzyme engineering
- enzyme selectivity
- ketoreductase
- protein structure
- substrate recognition
- substrate selectivity