Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency.
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.
Svensson
S
S
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Höög
J O
JO
Schneider
G
G
Sandalova
T
T
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D005559:Formamides / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006860:Hydrogen Bonding; D007850:Lauric Acids / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013379:Substrate Specificity
D001051:Apoenzymes; D005559:Formamides; D020035:Holoenzymes; D007850:Lauric Acids; D009243:NAD; C031263:12-hydroxydodecanoic acid; C105265:N-cyclohexylformamide; D006859:Hydrogen; D000426:Alcohol Dehydrogenase