title: Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.
abstract: The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). 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 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-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 favor 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 governed 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 ∼3.6 million.
authors.affiliation: Munich Center for Integrated Protein Science, CIPS-M, and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lerchner
authors.forename: Alexander
authors.initials: A
authors.lastname: Jarasch
authors.forename: Winfried
authors.initials: W
authors.lastname: Meining
authors.forename: André
authors.initials: A
authors.lastname: Schiefner
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D043367:Ralstonia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)
keywords: RasADH; Rossmann fold; enzymology; protein crystallography; protein engineering