Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

J Mol Biol. 2000 Sep 15;302(2):441-53. doi: 10.1006/jmbi.2000.4039.

Abstract

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / chemistry*
  • Alcohol Dehydrogenase / classification
  • Alcohol Dehydrogenase / genetics
  • Alcohol Dehydrogenase / metabolism*
  • Amino Acid Substitution / genetics
  • Animals
  • Apoenzymes / chemistry
  • Apoenzymes / genetics
  • Apoenzymes / isolation & purification
  • Apoenzymes / metabolism
  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Dimerization
  • Formamides / metabolism
  • Holoenzymes / chemistry
  • Holoenzymes / genetics
  • Holoenzymes / metabolism
  • Hydrogen / metabolism
  • Hydrogen Bonding
  • Lauric Acids / metabolism
  • Mice
  • Models, Molecular
  • Mutation / genetics
  • NAD / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Static Electricity
  • Substrate Specificity

Substances

  • Apoenzymes
  • Formamides
  • Holoenzymes
  • Lauric Acids
  • NAD
  • 12-hydroxydodecanoic acid
  • N-cyclohexylformamide
  • Hydrogen
  • Alcohol Dehydrogenase