Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase

Abstract

Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity.

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

“…C57 shows as light flipping, whereas W116 displays ac omplete flipping which indicatesa lso indirect effects of the mutation L119M ( Figure 6). Therefore, the binding of Me-(R)-3-OHC 4 (1b)i nacatalytic binding mode is enabled by conformationalc hanges enlarging the small binding pocket in the cpADH5 doublev ariant (L119M/W286S) which leads to af aster conversiona nd inversion of enantiopreference from S to R. For af ully quantitative prediction for enantiopreference of enzymatic reactions more sophisticated computational protocols including MD simulations and QM/MM calculations [23] are required to include also backboner earrengments and free energy contributions.W ang et al [22] observed as imilar shift of enantiopreference as an effect of the modification of residues in the smallb inding pocket of cpADH5. The increasei ns ize of the small binding pocket (W285A,W 286A,a nd W285A/W286A) showedashifti n enantiopreference for reduction of aryl ketones such as 3chloroacetophenone, 4-bromoacetophenone,a nd 4-chloro-2bromoacetophenone.…”

“…Both SSM libraries weres creened towardM e3-OHC 6 (1d)o xidation by spectrophotometrically monitoring of NADH production as previously reported. [22] 180 clones were screenedp er SSM positions( 95 %c overage [26] )a nd 1080clones for SSM double positions (83 %c overage;c alculated with GLUE-IT [26] ). Twenty variants with high activity toward Me3-OHC 6 (1d)w ere rescreened and the five most active variants were sequenced fore ach library.I na ll five cases an exchange from leucine to methionine was obtained at position L119, which is in agreement with previous reports about the conversion of bulky substrates like 2-methyl cyclohexanone.…”

“…The increasei ns ize of the small binding pocket (W285A,W 286A,a nd W285A/W286A) showedashifti n enantiopreference for reduction of aryl ketones such as 3chloroacetophenone, 4-bromoacetophenone,a nd 4-chloro-2bromoacetophenone. [22] Deng et al,i nverted the enantiopreferenceo fGluconobactero xydans carbonyl reductase toward ethyl 2-oxo-4-phenylbutyrate by mutating the active site resi- dues C93a nd Y149. Increased flexibility in the bindingp ocket resultedi nc ase of the substitutions C93P/Y149Ai na ni nversion of enantiopreference from R to S. [30] In order to investigate the effect of an enlarged binding pocket on the conversion of longer substrates, the activity of cpADH5t owardM e3-OHC 8 (1e)w as quantified.…”

“…pET22b-cpADH5, previously constructed, [20] was used as template for saturation mutagenesis. Positions 119a nd 286 were selected according to ap revious report [20,22] and computational analysis, respectively.F or generation of SSM libraries, am odified two step QuikChange Mutagenesis (QCM) protocol was applied. [31] In af irst step, two PCRs were performed in separate tubes;o ne containing the forward primer (F286-NNK for residue 286 and F119-NNK for residue 119) and the other containing the reverse primer (R286-NNK for residue 286 and R119-NNK for residue 119).…”

“…[16a] In addition, its substrate binding pocket was engineered throughs tructure guided design forr eduction of aryl ketones such as 2-bromo-4'-chloroacetophenone. [22] Recently,e nantioselectivity inversiono fThermoanaerobacter brockii secondary alcohold ehydrogenase (TbSADH) was explained through molecular dynamics simulations. [23] Engineering of proteins through rational or semi-rational approachese nables to tailor enzymep roperties such as substrate acceptance,t hermostability and enantiopreference.…”