X-ray structure of human class IV sigma sigma alcohol dehydrogenase - Structural basis for substrate specificity

The structural determinants of substrate recognition in the human class IV, or sigma sigma, alcohol dehydrogenase (ADH) isoenzyme were examined through x-ray crystallography and site-directed mutagenesis. The crystal structure of sigma sigma ADH complexed with NAD(+) and acetate was solved to 3-Angstrom resolution. The human beta(1) beta(1) and sigma sigma ADH isoenzymes share 69% sequence identity and exhibit dramatically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology within the sigma sigma substrate-binding pocket, relative to the beta(1) beta(1) isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the sigma sigma structure, appears to be twisted relative to its position in the beta(1) beta(1) isoenzyme. In conjunction with movements of Thr-48 and Phe-93, this twist widens the substrate pocket in the vicinity of the catalytic zinc and may contribute to this isoenzyme's high K-m for small substrates. The presence of Met-57, Met-141, and Phe-309 narrow the middle region of the sigma sigma substrate pocket and may explain the substantially decreased K-m values with increased chain length of substrates in sigma sigma ADH. The kinetic properties of a mutant sigma sigma enzyme (sigma 309L317A) suggest that widening the middle region of the substrate pocket increases K-m by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.