title: Protein engineering of a thermostable polyol dehydrogenase.
abstract: The polyol dehydrogenase PDH-11300 from Deinococcus geothermalis was cloned, functionally expressed in Escherichia coli and biochemically characterized. The enzyme showed the highest activity in the oxidation of xylitol and 1,2-hexanediol and had an optimum temperature of 45 °C. The enzyme exhibited a T⁶⁰₅₀-value of 48.3 °C. The T⁶⁰₅₀ is the temperature where 50% of the initial activity remains after incubation for 1h. In order to elucidate the structural reasons contributing to thermostability, the substrate-binding loop of PDH-11300 was substituted by the loop-region of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scope of this chimera basically represented the average of both wild-type enzymes, but surprisingly the T⁶⁰₅₀ was noticeably increased by 7 °C up to 55.3 °C. Further mutations in the active site led to identification of residues crucial for enzyme activity. The cofactor specificity was successfully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD⁺ binding cleft, without influencing the catalytic properties of the dehydrogenase.
authors.affiliation: Dept. of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str 4, D-17487 Greifswald, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Wulf
authors.forename: H
authors.initials: H
authors.lastname: Mallin
authors.forename: U T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006018:Glycols / Q000378:metabolism; D006586:Hexanes / Q000378:metabolism; D006358:Hot Temperature; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity; D014993:Xylitol / Q000378:metabolism
chemical_list: D006018:Glycols; D006586:Hexanes; D009243:NAD; D009249:NADP; D007064:L-Iditol 2-Dehydrogenase; C119102:1,2-hexanediol; D014993:Xylitol