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Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

ABSTRACT: The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.23, b = 124.90, c = 164.80 A. The structure was solved by molecular replacement using the structure of T. brockii SADH (PDB entry 1ykf).

SUBMITTER: Protsko C

PROVIDER: S-EPMC2898473 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

Protsko Carla C Vieille Claire C Laivenieks Maris M Prasad Lata L Sanders David A R DA Delbaere Louis T J LT

Acta crystallographica. Section F, Structural biology and crystallization communications 20100624 Pt 7

The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.23, b = 124.90, c = 164.80 A. The structure was solved by molecular replacement using the st ...[more]

PMID: 20606285

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Project description:The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had </= 3% of wild-type catalytic activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH,the Rossmann-fold-containing T. ethanolicus secondary ADH binds its catalytic Zn atom using a sorbitol dehydrogenase-like Cys-His-Asp motif and does not bind a structural Zn atom.

Dataset Information

Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

Publications

Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

Similar Datasets

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