Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability

Abstract

The crystal structure of a thermophilic alcohol dehydrogenase (TBAD) from Thermoanaerobacter brockii has been determined in a binary complex with sec-butanol as substrate to a resolution of 3.0 A. Van der Waals interactions of the carbon C1 atom of sec-butanol with atoms in His59, Ala85, Trp110, Asp150, and Leu294 account for the substrate preference of this enzyme for secondary over primary alcohols. A crevice from the surface to the active site provides access for substrates and products. This opening is lined with the hydrophobic residues Ile49, Leu107, Trp110, Tyr267, Leu294 as well as Cys283 and Met285 from another molecule within the tetrameric assembly. This might explain the tolerance of this enzyme toward organic solvents. The zinc ion occupies a position in the active site, which is too remote for direct interaction with the alcohol group. A mechanism is suggested whereby the introduction of NADP would trigger a displacement of the zinc ion to its catalytic site. Features important for the unusually high melting temperature of 98 degrees C are suggested by comparison to the crystal structure of a highly homologous mesophilic alcohol dehydrogenase from Clostridium beijerinckii (CBAD). The thermophilic enzyme has a more hydrophilic exterior, a more hydrophobic interior, a smaller surface area, more prolines, alanines, and fewer serines than CBAD. Furthermore, in the thermophilic enzyme the number of all types of intersubunit interactions in these tetrameric enzymes is increased: more salt bridges, hydrogen bonds, and hydrophobic interactions. All these effects combined can account for the higher melting temperature of the thermophilic enzyme.

Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster

“…The major proteins with a native alcohol-binding function are alcohol dehydrogenases, which invariably involve coordination of metal ions and other cofactors to the alcohol to perform an enzymatic reaction [14][15][16][17] . This mode of alcohol binding is unrelated to the non-enzymatic sites that occur in alcohol-sensitive ion-channels.…”

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

“…This is remarkable, since the original host, C. beijerinckii NCIMB 8052, is a mesophilic microorganism. However, it has been observed more often that ADHs from C. beijerinckii are relatively stable at moderate temperatures (30)(31)(32). Also, closely related thermophilic Clostridium species are known to possess proteins with activity optima at higher temperatures (33).…”

Structural Basis for Thermostability of β-Glycosidase from the Thermophilic Eubacterium Thermus nonproteolyticus HG102

“…Replacement of other residues by Pro at suitable positions can enhance protein thermostability (19). It has been noted that Pro has an increased occurrence in thermophilic proteins, especially in loops (5,30,52). The Pro content in Gly Tn (8.0%) is the highest of all nine glycosidases, and the positional distribution in Gly Tn of the total 35 Pro residues was examined.…”

“…Some of the families can be grouped into "clans," because the folds of their proteins are better conserved than their sequences (23). Families 1,2,5,10,17,26,30,35,39,42,51,53,59, 72, 79, and 86 are grouped into a superfamily, clan GH-A. Members of this superfamily adopt a (␤␣) 8 barrel fold (23).…”