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Publication Abstract from PubMed

The NADP+ -dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases. As an enzyme that accepts different types of substrates - including bulky-bulky as well as small-bulky secondary alcohols or ketones - with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP+ -bound state with resolutions down to 2.8 A. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel beta-sheet, flanked by three alpha-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favour practical RasADH application in the context of established cofactor recycling systems, for example those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP+ to NAD+ utilizing the structural information that NADP+ specificity is largely conferred by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S) and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH(N15G/G37D/R38V/R39S) was better able to accept NAD+ while showing much reduced catalytic efficiency with NADP+ , leading to a change in NADH/NADPH specificity by a factor of approximately 3.6 million. Biotechnol. Bioeng. (c) 2013 Wiley Periodicals, Inc.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.,Lerchner A, Jarasch A, Meining W, Schiefner A, Skerra A Biotechnol Bioeng. 2013 May 18. doi: 10.1002/bit.24956. PMID:23686719^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.