Maria Krook

Short-chain dehydrogenases/reductases (SDR).

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

The primary structure of 20β‐hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation. Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC. The 255‐residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20β‐hydroxysteroid dehydrogenase as a member of the ‘short‐chain alcohol dehydrogenase family’. With the enzymes characterized, the identity is greatest (31–34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases. These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important.

Three-dimensional model of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase and relationships to the NADP+-dependent enzyme (carbonyl reductase)

Modelling the amino acid sequence of NAD+‐linked 15‐hydroxyprostaglandin dehydrogenase into the three‐dimensional structure of 3α/20β‐hydroxysteroid dehydrogenase shows that these two enzymes, as well as the NADP+‐linked prostaglandin dehydrogenase (identical to carbonyl reductase) have similar conformations, in spite of very limited sequence identity (23–28%). Conservation of tertiary structures is greatest over the first two thirds of the polypeptide chains, where the typical NAD+ binding fold is retained, including the five first β‐strands, with only two short deletions or insertions up to residue 147. The remaining thirds of each of the prostaglandin dehydrogenases have significantly different architecture, including insertions that may contribute to enzyme specificity, and, except for an additional helix (αG), are difficult to model. Active site relationships can be evaluated and subunit interactions predicted, suggesting that the αE + αF two‐helix surface constitutes the major subunit interacting area, forming a dimeric unit in the oligomeric enzymes.

Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr‐109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase. Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super‐activation with cyanide was also noticed after S‐sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.

C1pB proteins copurify with the anaerobic escherichia coli reductase

Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3314-3318). Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease. The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase.