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Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity.
J Mol Biol. 2005 Jun 17; 349(4):801-13.JM

Abstract

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.

Authors+Show Affiliations

Universität zu Köln, Institut für Biochemie, Zülpicher Strasse 47, D-50674 Köln, Germany.No affiliation info availableNo 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

15896805

Citation

Schlieben, Nils Helge, et al. "Atomic Resolution Structures of R-specific Alcohol Dehydrogenase From Lactobacillus Brevis Provide the Structural Bases of Its Substrate and Cosubstrate Specificity." Journal of Molecular Biology, vol. 349, no. 4, 2005, pp. 801-13.
Schlieben NH, Niefind K, Müller J, et al. Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity. J Mol Biol. 2005;349(4):801-13.
Schlieben, N. H., Niefind, K., Müller, J., Riebel, B., Hummel, W., & Schomburg, D. (2005). Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity. Journal of Molecular Biology, 349(4), 801-13.
Schlieben NH, et al. Atomic Resolution Structures of R-specific Alcohol Dehydrogenase From Lactobacillus Brevis Provide the Structural Bases of Its Substrate and Cosubstrate Specificity. J Mol Biol. 2005 Jun 17;349(4):801-13. PubMed PMID: 15896805.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity. AU - Schlieben,Nils Helge, AU - Niefind,Karsten, AU - Müller,Jörg, AU - Riebel,Bettina, AU - Hummel,Werner, AU - Schomburg,Dietmar, PY - 2004/12/30/received PY - 2005/04/08/revised PY - 2005/04/14/accepted PY - 2005/5/18/pubmed PY - 2005/7/13/medline PY - 2005/5/18/entrez SP - 801 EP - 13 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 349 IS - 4 N2 - The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/15896805/Atomic_resolution_structures_of_R_specific_alcohol_dehydrogenase_from_Lactobacillus_brevis_provide_the_structural_bases_of_its_substrate_and_cosubstrate_specificity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(05)00438-9 DB - PRIME DP - Unbound Medicine ER -