Crystal structure of cod liver class I alcohol dehydrogenase: Substrate pocket and structurally variable segments

Abstract

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops.Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class 1 enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6". In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.

X-ray Structure of Human Class IV ςς Alcohol Dehydrogenase

“…The K m value for NAD ϩ was approximately 2 times higher than the wild-type enzyme, whereas the K i (NAD C␣ alignment of ADH with horse and other human ADHs shows that its structure is most similar to the human and horse liver class I ADH isoenzymes. Unlike the recently reported structures of the human class III isoenzyme (13) and the ADH isoenzyme from cod liver (14), both of which exhibited semi-open domain structures, the human isoenzyme exhibits a fully closed conformation of the catalytic and coenzyme-binding domains when NAD(H) is bound. The alignments reveal that, relative to ␤ 1 , there are two major structural differences in each domain of the subunit (Fig.…”

“…Recently, the structure of human class III ADH was reported (13), as well as the structure of a cod liver ADH isoenzyme (14). Thus, an increasingly diverse structural data base exists from which information concerning the determinants of substrate recognition can be obtained by comparing the structures and kinetic properties of ADH isoenzymes.…”

Mycothiol‐Dependent Formaldehyde Dehydrogenase, A Prokaryotic Medium‐Chain Dehydrogenase/Reductase, Phylogenetically Links Different Eukaroytic Alcohol Dehydrogenases Primary Structure, Conformational Modelling and Functional Correlations

“…2). The position of these internal insertions and deletions correspond to a superficial loop in the tertiary structure of those MDR enzymes that have been crystallographically analyzed, the horse class-I [ 211, human class-I [22], and cod class-1411 hybrid [24] [35], has been ascribed to a more complex quateriiary structure than that of the dimeric mammalian liver forms, which have the loop. Consequently, the present structure was submitted to molecular modelling starting from the human class-I alcohol dehydrogenase to evaluate the loop structure and other segments.…”

A Novel Subtype of Class II Alcohol Dehydrogenase in Rodents

“…The conserved His 51 in class I ADHs acts as a general base catalyst through a proton relay connecting the alcohol substrate and His 51 via the hydroxyl group of Thr/Ser 48 and the 2Ј-hydroxyl of the nicotinamide ribose (48 -52). Structures of binary complexes of both cod ADH and human class III ADH show that His 47 is at a position where hydrogen binding to the 2Ј-hydroxyl is possible and could therefore potentially act in the same manner as His 51 (53,54). In addition, the drop in activity seen for a His for Gln mutant at a position analogous to 47 of Pseudomonas putida benzyl alcohol dehydrogenase could partially be restored by introduction of either exogenous amines as proton acceptors or His at a position analogous to 51 (27).…”