title: The three-dimensional structure of AKR11B4, a glycerol dehydrogenase from Gluconobacter oxydans, reveals a tryptophan residue as an accelerator of reaction turnover.
abstract: The NADP-dependent glycerol dehydrogenase (EC 1.1.1.72) from Gluconobacter oxydans is a member of family 11 of the aldo-keto reductase (AKR) enzyme superfamily; according to the systematic nomenclature within the AKR superfamily, the term AKR11B4 has been assigned to the enzyme. AKR11B4 is a biotechnologically attractive enzyme because of its broad substrate spectrum, combined with its distinctive regioselectivity and stereoselectivity. These features can be partially rationalized based on a 2-Å crystal structure of apo-AKR11B4, which we describe and interpret here against the functional complex structures of other members of family 11 of the AKR superfamily. The structure of AKR11B4 shows the AKR-typical (β/α)(8) TIM-barrel fold, with three loops and the C-terminal tail determining the particular enzymatic properties. In comparison to AKR11B1 (its closest AKR relative), AKR11B4 has a relatively broad binding cleft for the cosubstrate NADP/NADPH. In the crystalline environment, it is completely blocked by the C-terminal segment of a neighboring protomer. The structure reveals a conspicuous tryptophan residue (Trp23) that has to adopt an unconventional and strained side-chain conformation to permit cosubstrate binding. We predict and confirm by site-directed mutagenesis that Trp23 is an accelerator of (co)substrate turnover. Furthermore, we show that, simultaneously, this tryptophan residue is a critical determinant for substrate binding by the enzyme, while enantioselectivity is probably governed by a methionine residue within the C-terminal tail. We present structural reasons for these notions based on ternary complex models of AKR11B4, NADP, and either octanal, d-glyceraldehyde, or l-glyceraldehyde.
authors.affiliation: Evocatal GmbH, Merowingerplatz 1A, D-40225 Düsseldorf, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Richter
authors.forename: Klaus
authors.initials: K
authors.lastname: Breicha
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
authors.forename: Karsten
authors.initials: K
authors.lastname: Niefind
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020240:Apraxia, Ideomotor; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009249:NADP; D014364:Tryptophan; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase