Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759 bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781 Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558 U mg−1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH 7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the c...

Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

https://pubs.rsc.org/en/Content/ArticlePDF/2018/CC/C8CC02426J

Role of conformational dynamics in the evolution of novel enzyme function

The biocatalysis discipline has the potential to generate important chiral organic compounds by the use of enzymes, usually in the presence of critical co-factors. Therefore, practical methods for the regeneration of the co-enzyme, 1,4-NADH, have continued to be of significance in the biocatalysis field. In this vain, a variety of transition metal hydrides have been evaluated as catalysts for the regioselective reduction of NAD+ and NAD+ models to the corresponding 1,4-NADH derivatives in order to attempt to develop faster rates and a more economical regeneration process. In the most significant example, Steckhan and co-workers have described the use of in situ generated [Cp*Rh(bpy)(H)]+ (Cp* = η-C5Me5, bpy = 2,2'-bipyridyl), for the regiospecific reduction of natural NAD+ to 1,4-NADH, and then demonstrated the co-factor regeneration process in

/pdf/biomimetic-nad-models-for-tandem-cofactor-regeneration-horse-3z21wsw6he.pdf

Biomimetic NAD(+) models for tandem cofactor regeneration, horse liver alcohol dehydrogenase recognition of 1,4-NADH derivatives, and chiral synthesis.

Zinc-dependent enzymes play important roles in many cellular processes Assignment of their reaction mechanisms is often a subject of debate because the zinc ion is silent in several spectroscopic techniques We have combined time-resolved X-ray absorption spectroscopy, pre-steady state kinetics and computational quantum chemistry to study the active site zinc ion of bacterial alcohol dehydrogenase during single substrate turnover We detect a series of alternations in the coordination number and structure of the catalytic zinc ion with concomitant changes in metal–ligand bond distances These structural changes are reflected in the effective charge of the metal ion The present work emphasizes the flexibility of catalytic zinc sites during catalysis and provides novel mechanistic insights into alcohol dehydrogenase catalysis

Active site electronic structure and dynamics during metalloenzyme catalysis.

Crystal structure of the alcohol dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus at 1.85 A resolution.

Thermoanaerobacter brockiialcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP + as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state.

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Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state.