Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases

Abstract

This is the accepted version of a paper published in ACS Catalysis. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.

Evolutionary Effects on Bound Substrate pKa in Dihydrofolate Reductase

“…The current findings regarding evolutionarily controlled substrate p K a in enzymes present a fundamental advance in our understanding of enzymes. The fact that enzymes have evolved to control substrate p K a can possibly aid in site directed mutagenesis (e.g., directed evolution) to engineer evolvable enzymes that are more efficient, with improved catalytic rates and serve as catalysts for unnatural substrates/reactions. − …”

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

“…substrate scope ISM C3-epimerization of d-fructose and l-sorbose [126] Cyclodextrin Glycosyltransferase (Bacillus stearothermophilus NO2) activity ISM ISM on nine residues in the binding pocket [127] ADH-A from Rhodococcus ruber DSM 44 541 stereo-and regioselectivity ISM ISM combinedw ith activity screening [128] approaches was application of the Sloning technique of solidphase gene synthesis,i nw hich we compared ac onventional SM library with the Sloning counterpart. [145] TheS loning library appeared to have ah igher quality,b ut at the time we neglected to perform massive DNAs equencing for reliable assessment.…”

The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase

“…The volumes of the active site cavities of the ADH-A variants considered in this work were analyzed by CASTp 3.0, using the CASTp online server (). The analysis was specifically performed on PDB IDs: 3jv7 (wild-type ADH-A), 5o9d (A2C2B1 variant), 5o9f (A2C3 variant), 5od3 (B1 variant), 6fg0 (B1F4 variant), 6ffx (C1 variant), and 6ffz (C1B1 variant). − , The A2 variant crystallized into a different space group ( P 2 1 2 1 2 1 ) as compared with all the other variants (P2 1 ), and the asymmetric unit cell contained eight subunit chains rather than four. Therefore, to lower the computational cost of the analysis and to mimic the oligomeric state of the other proteins in the crystalline state, the coordinates file for the A2 variant (PDB ID: 5o8q ) was truncated to contain four subunit chains before analysis.…”

“…Partial charges were calculated at the HF/6-31G* level of theory, using Gaussian09, and then fitted using the standard RESP procotol (Table S2). The parameters for NAD + were taken from the literature. , We then used a tetrahedral cationic dummy model to describe the two divalent zinc ions per subunit on the basis of previous successful results using this model . In order to prevent the nearby side chain of E63 from artificially coordinating with the metal ion, thus displacing the metal-coordinating H62 side chain, the charges of the H62 side chain were modified on the basis of examining the charge distributions obtained from quantum mechanical calculations of 4-methylimidizaole tetrahedrally coordinated to a zinc metal center, with the coordination shell completed by three extra water molecules.…”

“…We have previously studied and engineered alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 − (Figure ) for the purpose of generating new enzyme forms with predesigned catalytic properties regarding substrate scope and selectivity. − …”