Mustafa El-Ahmad

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A-B and C-D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.

Alcohol Dehydrogenase Variability

We have studied many alcohol dehydrogenases and related enzymes with the aim of defining functional properties, structural patterns, and evolutionary relationships. From this, four major conclusions have been drawn: The enzymes are clearly multiple and represent different protein families. Within the MDR family (medium-chain dehydrogenases/reductases), repeated duplications at different levels have produced the enzymes, classes, and isozymes that are now visible in human, mammalian, and other lines (Jornvall et al., 1987; Hjelmqvist et al., 1995a). Of the alcohol dehydrogenases, class III, with its glutathione-dependent formaldehyde dehydrogenase activity (Koivusalo et al., 1989), appears to be the parent form, locking much of the alcohol dehydrogenase family to cellular detoxication reactions (Danielsson and Jornvall, 1992). Separate, internal molecular architectures are present (Danielsson et al., 1994a). Class III has a protein-classical pattern, with a low variability overall like functionally constant enzymes in general, and with variable regions in non-functional segmens. The other classes are more variable, both overall and in their functional segments, in a protein-atypical manner, indicating evolution of new functions, or “enzymogenesis” (Danielsson and Jornvall, 1992). Functional convergence toward ethanol activity has occurred in many lines. Thus, the ethanol-active enzymes in yeast, prokaryotes, plants, and animals all appear to have separate origins (Jornvall, 1994).

Crystallisation and crystallographic investigations of cod alcohol dehydrogenase class I and class III enzymes.

Cod liver alcohol dehydrogenase of class‐hybrid properties has been crystallized as an NAD+—pyrazole complex in the monoclinic space group P21 with cell dimensions a = 103.3 Å, b = 47.4 Å, c = 80.7 Å, β = 104.6°, and with one dimer in the asymmetric unit. The position of the dimer molecule in the crystal was determined by molecular replacement methods at 3.0 Å resolution. The successful search model was the poly‐alanine structure of the horse enzyme. Side chains were then replaced according to the amino acid sequence of the cod enzyme, and the structure has been refined at 2.8 Å to an R‐factor of 0.26. Cod liver class III alcohol dehydrogenase crystallizes in the monoclinic space group C2 with cell dimensions a = 127.5 Å, b = 76.6 Å, c = 93.4 Å, β = 99.4° and with probably one dimer in the asymmetric unit.

Class III alcohol dehydrogenase: consistent pattern complemented with the mushroom enzyme

Mushroom alcohol dehydrogenase (ADH) from Agaricus bisporus (common mushroom, champignon) was purified to apparent homogeneity. One set of ADH isozymes was found, with specificity against formaldehyde/glutathione. It had two highly similar subunits arranged in a three‐member isozyme set of dimers with indistinguishable activity. Determination of the primary structure by a combination of chemical, mass spectrometric and cDNA sequence analyses, correlated with molecular modeling towards human ADHs, showed that the active site residues are of class III ADH type, and that the subunit differences affect other residues. Class I and III forms of ADHs characterized define conserved substrate‐binding residues (three and eight, respectively) useful for recognition of these enzymes in any organism.

Crystallographic investigations of alcohol dehydrogenases

The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.

Structure and Function of Betaine Aldehyde Dehydrogenase

Aldehyde and alcohol dehydrogenases (ALDHs and ADHs) are both highly multiple enzymes with many different forms in metabolically linked functional pathways. However, they are derived from separate protein families with distinct properties. Those of the vertebrate ADHs have been well characterized since long, and their different classes belong to the MDR protein family of known structures from several sources. However, the properties and multiplicity of the ALDHs have only recently been partly characterized. Presently, just three of the at least twelve different ALDH classes (Yoshida et al., 1998) are kn in three-dimensional structure (Liu et al., 1997; Steinmetz et al., 1997; Johansson et al.998), several have not been purified and characterized enzymatically, and little is known about their species or class variabilities.

Crystallizations of Novel Forms of Alcohol Dehydrogenase

Alcohol dehydrogenases in nature are derived from different protein families (cf Jornvall et al., 1993). The mammalian alcohol dehydrogenases constitute what presently appears to be six classes within a large family of medium-chain dehydrogenases and reductases, MDR, with at least seven characterized activity types (Persson et al., 1994). The classes of the mammalian alcohol dehydrogenases differ in structure (55–68% sequence identity), substrate pockets, subunit interactions, and other properties. Much of these aspects has been interpreted by comparisons and modelling studies based on crystallographic analyses of just two of the enzymes of one class, the class I horse (Eklund et al., 1976) and human (Hurley et al., 1991) enzymes, constituting the classical liver enzyme with ethanol activity. It is desirable to get direct crystallographic data on the conformations of more of the enzymes, especially since differences exist between the classes, class-hybrid properties have been found in early enzyme forms (of fish), and the two well-characterized classes, I and III, differ in internal variability patterns (Danielsson et al., 1994a). We have therefore crystallized five novel forms of these dehydrogenases, which should enable further structural characterizations and hence evaluation of the conclusions from modelling and from natural variants.