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.

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Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.

Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "shortchain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and verte-brates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class m enzyme, consistent with an important role for this enzyme in cellular metabolism.The "classical" alcohol dehydrogenase is part of a widespread system of zinc-containing enzymes (1). In mammalian tissues, at least six classes of this enzyme occur. They differ considerably and represent stages between separate enzymes and ordinary isozymes. Class I is the well-known liver enzyme with ethanol dehydrogenase activity (2), class III is identical with glutathione-dependent formaldehyde dehydrogenase (3), class IV is a form preferentially expressed in stomach (4, 5), while classes II, V, and VI, although little studied, are known also to exhibit distinct properties (6, 7, 44). The class origins have been traced to gene duplications early in vertebrate evolution [the I/III duplication (8)] or during that evolution [the IV/I duplication (5)], with emerging activities toward ethanol (9); class III corresponds to an ancestral form. These properties and the different evolutionary patterns, with class III being "constant" and class I "variable" (10), result in a consistent picture of the enzyme system and place the classes of medium-chain alcohol dehydrogenases as separate enzymes in the cellular metabolism.Similarly, another protein family, short-chain dehydrogenases, has also evolved into a family comprising many different enzyme activities, including an alcohol dehydrogenase (11). This form operates by means of a completely different catalytic mechanism and is related to mammalian prostaglandin dehydrogenases/carbonyl reductase (12). Thus far, this alcohol dehydrogenase has been found in insects, the Drosophila enzyme being recognized early to differ from the zinc-containing alcohol dehydrogenases (13,14). Its properties in various Drosophila species are well established (15).These two alcohol dehydrogenase types demonstrate that ethanol dehydrogenase activity has evolved in different manners, with many organisms now employing a medium-chain enzyme, while others depend on a short-chain enzyme. The medium-chain family has not been identified in insects, although it is of ancient origin and has been characterized in other eukaryotes and in prokaryotes. We now show that the family is indeed present also in insects and that its major representative is the typical class III t...

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Development of many chordate features depends on retinoic acid (RA). Because the action of RA during development seems to be restricted to chordates, it had been previously proposed that the "invention" of RA genetic machinery, including RA-binding nuclear hormone receptors (Rars), and the RA-synthesizing and RA-degrading enzymes Aldh1a (Raldh) and Cyp26, respectively, was an important step for the origin of developmental mechanisms leading to the chordate body plan. We tested this hypothesis by conducting an exhaustive survey of the RA machinery in genomic databases for twelve deuterostomes. We reconstructed the evolution of these genes in deuterostomes and showed for the first time that RA genetic machinery--that is Aldh1a, Cyp26, and Rar orthologs--is present in nonchordate deuterostomes. This finding implies that RA genetic machinery was already present during early deuterostome evolution, and therefore, is not a chordate innovation. This new evolutionary viewpoint argues against the hypothesis that the acquisition of gene families underlying RA metabolism and signaling was a key event for the origin of chordates. We propose a new hypothesis in which lineage-specific duplication and loss of RA machinery genes could be related to the morphological radiation of deuterostomes.

Genetic engineering, coupled with spectroscopic analyses, has enabled the metal binding properties of the alpha and beta subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn4-alpha MT and Zn3-beta MT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to alpha fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the native protein. Titration of Zn3-beta MT with CdCl2 results in the formation of Cd3-beta MT. The addition of excess Cd(II) leads to Cd4-beta MT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-beta MT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd3-beta MT species is stable in the presence of this strong metal-chelating agent.