Jonathan Jeffery

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.

The primary prostaglandin-inactivating enzyme of human placenta is a dimeric short-chain dehydrogenase

The native form of NAD-dependent 15-hydroxyprostaglandin dehydrogenase of human placenta has a mol. wt. of about 50 0002 while the subunit tool. wt. is around 2g 0002 suggesting a dimeric quaternary structure. These propertie% the amino acid composition, insensitivity to EDTA, and inhibition patterns show general similarities to other short-chain dehydrogenases. Several hormones tested did not influence the activity of 15-hydroxyprostaglandin dehydrogenase2 but an unusual activation by two anti-depressant drugs was found and may relate to the existence of a natural regulatory factor.

Different segment similarities in long-chain dehydrogenases

Long-chain dehydrogenases were scrutinized for common patterns. Overall molecular similarities are not discerned, in contrast to the situation for several short-chain and medium-chain dehydrogenases, but coenzyme-binding segments are discernible. Species variants of glucose-6-phosphate dehydrogenase reveal about 20% strictly conserved residues, grouped into three segments and supporting assignments of sites for coenzyme-binding and catalysis. Glycine is overrepresented among the residues conserved, typical of distantly related proteins. Two of the enzymes within the pentose phosphate pathway reveal a distant similarity of interest for further evaluation, between a C-terminal 178-residue segment of glucose-6-phosphate dehydrogenase and the N-terminal part of 6-phosphogluconate dehydrogenase.

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis, Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.

Steric, chiral and conformational aspects of the 3-hydroxy- and 20-hydroxysteroid dehydrogenase activities of cortisone reductase preparations

Abstract The 3-hydroxysteroid dehydrogenase activity of cortisone reductase (20-dihydrocortisone:NAD+ oxidoreductase, EC 1.1.1.53) was known to be 3α activity in the case of certain 5α-steroids in which 3α was 3R and axial. It is now shown to be 3α activity also in a 5α-steroid in which 3α is 3S and axial. In the 5β series, both 3α, 3R, equatorial activity and 3β, 3S axial activity are now described, though the compounds were very poor substrates. The view is discussed that interactions between the all-trans-5α-steroids and the enzyme distinguish the α side of the steroid from the β side; and that the angled A/Bcis-steroids of the 5β series are either bound somewhat differently, or are possibly bound in more than one way. The 20-hydroxysteroid dehydrogenase activity of cortisone reductase was known to be 20β activity in various cases where 20β was 20R. It is now shown to be 20β activity also in a case where 20β is 20S. In the case of 20 activity, the orientation around C-20 at the moment of hydrogen transfer is evidently fixed relative to both the steroid nucleus and the hydrogen donor or acceptor (presumably enzyme-bound NADH or NAD+) even though rotation about the C-17/C-20 bond is possible.

Glucose-6-phosphate dehydrogenase. Characterization of a reactive lysine residue in the Pichia jadinii enzyme reveals a limited structural variation in a functionally significant segment.

Glucose-6-phosphate dehydrogenase from the yeast Pichia jadinii has a reactive lysine residue in a segment of amino acid sequence Ile-Asp-His-Tyr-Leu-Gly-Lys*-Glu-Met-Val-Lys. This structure differs from that of other characterized glucose-6-phosphate dehydrogenases, but outside yeasts the segment is invariant in known mammalian, insect and bacterial forms. Thus, limited structural variation is now defined within yeasts for a part of the protein otherwise strictly conserved, and for which stringent structural requirements probably relate to enzymic mechanisms.

Structural features of ring B and the interaction of 20-oxosteroids with cortisone reductase.

Abstract Kinetic measurements were made using cortisone reductase (20-dihydrocortisone: NAD + -oxidoreductase, EC. 1.1.1.53) and a series of substrates which differed in shape, size, and electronic character in the region around C-6, C-7 and C-10. These structural changes in the substrates resulted in up to 120-fold changes in the apparent K m , up to 14-fold changes in the apparent V , and up to 380-fold changes in the ratio of these kinetic constants. It is suggested that interactions important, but not essential, for substrate function normally occur between the enzyme and the region around ring B, and that these include hydrophobic interactions with the β-side of ring B.

Eukaryotic glucose-6-phosphate dehydrogenases : structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.

Chapter 3 Stereochemistry of dehydrogenases

Publisher Summary Dehydrogenases (or oxidoreductases) constitute the first of six main divisions in the Enzyme Commission classification. About 300 dehydrogenases that utilize nicotinamide coenzymes are known, and this chapter deals with some of them. The recommended name and EC number designate “not a single enzyme protein, but a group of proteins with the same catalytic property. General properties of polypeptide chains cause formation of α-helices and β-sheets. The combination of these secondary structures in dehydrogenases leads to tertiary structures that are not uniform, but which show striking similarities, for example in the coenzyme binding domains of NAD-dependent dehydrogenases (Rossmann folds). The gross structural characteristics of dehydrogenases therefore seem to derive from general properties of polypeptide chains, and it is found that similar tertiary structures have resulted from considerably different amino acid sequences. The steric course of reaction is determined by relatively non-specific interactions, such as van der Waals contacts that would not allow a bulky group to be accommodated the other way round, and by specific interactions such as hydrogen bonds.