Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity.
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.
Schlieben
Nils Helge
NH
Universität zu Köln, Institut für Biochemie, Zülpicher Strasse 47, D-50674 Köln, Germany.
Niefind
Karsten
K
Müller
Jörg
J
Riebel
Bettina
B
Hummel
Werner
W
Schomburg
Dietmar
D
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003067:Coenzymes; D000426:Alcohol Dehydrogenase; D005998:Glycine