Structural Basis for Branched Substrate Selectivity in a Ketoreductase from Ascaris suum

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.

Discovery and Process Development of a Scalable Biocatalytic Kinetic Resolution toward Synthesis of a Sterically Hindered Chiral Ketone

“…To further evaluate the performance of KRED 119, we conducted a series of presteady state Michaelis–Menten kinetics studies (Scheme ). Key results suggest that the binding affinity of ketone 1 to the chosen enzyme ( K m ) and its turnover number ( k cat ) are moderate, delivering the catalytic efficiency as 1.3 × 10 4 M –1 s –1 . These results are not surprising, given that the selected reaction is not native to this wild-type enzyme.…”

Cancer to Cataracts: The Mechanistic Impact of Aldo-Keto Reductases in Chronic Diseases

“…AKR1B1 also reduces many AKR1A1 substrates but with less activity. However, this is untrue as ketoreductases with larger substrate sites are better at catalyzing aromatic ketones compared to aliphatic ketones [ 93 , 94 ]. However, this notion is hypothetical at best as AKRs have not been extensively compared against the same substrates and warrants further exploration.…”