4I5F

Crystal structure of Ralstonia sp. alcohol dehydrogenase mutant N15G, G37D, R38V, R39S

Summary for 4I5F

• Entry DOI: 10.2210/pdb4i5f/pdb
• Related: 4I5D 4I5E 4I5G
• Descriptor: Alclohol dehydrogenase/short-chain dehydrogenase (2 entities in total)
• Functional Keywords: short-chain-dehydrogenases/reductases, rossmann fold, ralstonia sp., alcohol dehydrogenase, rasadh, cosubstrate specificity, nadh, s-phenylethanol, oxidoreductase
• Biological source: Ralstonia sp.
• Total number of polymer chains: 8
• Total formula weight: 224405.34

Authors

Jarasch, A.,Lerchner, A.,Meining, W.,Schiefner, A.,Skerra, A. (deposition date: 2012-11-28, release date: 2013-06-12, Last modification date: 2023-09-20)

Primary citation

Lerchner, A.,Jarasch, A.,Meining, W.,Schiefner, A.,Skerra, A.
Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. Alcohol dehydrogenase toward NADH cosubstrate specificity.
Biotechnol.Bioeng., 110:2803-2814, 2013

PubMed 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.

PubMed: 23686719
DOI: 10.1002/bit.24956

Experimental method

X-RAY DIFFRACTION (2.1 Å)