Tags

Type your tag names separated by a space and hit enter

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.
Biotechnol Bioeng. 2013 Nov; 110(11):2803-14.BB

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+Show Affiliations

Munich Center for Integrated Protein Science, CIPS-M, and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23686719

Citation

Lerchner, Alexandra, et al. "Crystallographic Analysis and Structure-guided Engineering of NADPH-dependent Ralstonia Sp. Alcohol Dehydrogenase Toward NADH Cosubstrate Specificity." Biotechnology and Bioengineering, vol. 110, no. 11, 2013, pp. 2803-14.
Lerchner A, Jarasch A, Meining W, et al. Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity. Biotechnol Bioeng. 2013;110(11):2803-14.
Lerchner, A., Jarasch, A., Meining, W., Schiefner, A., & Skerra, A. (2013). Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity. Biotechnology and Bioengineering, 110(11), 2803-14. https://doi.org/10.1002/bit.24956
Lerchner A, et al. Crystallographic Analysis and Structure-guided Engineering of NADPH-dependent Ralstonia Sp. Alcohol Dehydrogenase Toward NADH Cosubstrate Specificity. Biotechnol Bioeng. 2013;110(11):2803-14. PubMed PMID: 23686719.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity. AU - Lerchner,Alexandra, AU - Jarasch,Alexander, AU - Meining,Winfried, AU - Schiefner,André, AU - Skerra,Arne, Y1 - 2013/07/01/ PY - 2013/02/14/received PY - 2013/04/21/revised PY - 2013/04/29/accepted PY - 2013/5/21/entrez PY - 2013/5/21/pubmed PY - 2014/4/5/medline KW - RasADH KW - Rossmann fold KW - enzymology KW - protein crystallography KW - protein engineering SP - 2803 EP - 14 JF - Biotechnology and bioengineering JO - Biotechnol. Bioeng. VL - 110 IS - 11 N2 - 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. SN - 1097-0290 UR - https://www.unboundmedicine.com/medline/citation/23686719/Crystallographic_analysis_and_structure_guided_engineering_of_NADPH_dependent_Ralstonia_sp__alcohol_dehydrogenase_toward_NADH_cosubstrate_specificity_ L2 - https://doi.org/10.1002/bit.24956 DB - PRIME DP - Unbound Medicine ER -