Physicochemical characterization of a thermostable alcohol dehydrogenase from Pyrobaculum aerophilum.

Methods Development

Abstract

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.

Authors

Vitale, Annalisa; Thorne, Natasha; Lovell, Scott; Battaile, Kevin P; Hu, Xin; Shen, Min; D'Auria, Sabato; Auld, Douglas S;

Keywords

  • Alcohol Dehydrogenase/ antagonists & inhibitors
  • Alcohol Dehydrogenase/ chemistry
  • Apoenzymes/ antagonists & inhibitors
  • Apoenzymes/ chemistry
  • Archaeal Proteins/ antagonists & inhibitors
  • Archaeal Proteins/ chemistry
  • Biocatalysis
  • Catalytic Domain
  • Cobalt/ chemistry
  • Coordination Complexes/ chemistry
  • Crystallography, X-Ray
  • Enzyme Assays
  • Enzyme Stability
  • Flunarizine/ chemistry
  • High-Throughput Screening Assays
  • Hydrogen Bonding
  • Models, Molecular
  • NADP/ chemistry
  • Protein Structure, Secondary
  • Pyrobaculum/ enzymology
  • Structural Homology, Protein
  • Tetralones/ chemistry
  • Zinc/ chemistry

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