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.
Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17P-hydroxysteroid and 11 P-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues, Asp64, Tyr152 and Lysl56 (in the numbering of Drosophilu alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyrl52 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.Several highly different types of alcohol dehydrogenase and related enzymes are known [l -41. The short-chain dehydrogenase family (subunits with about 250 residues) was initially discerned from insect alcohol dehydrogenase and bacterial ribitol dehydrogenase [I, 5-71. Knowledge about this family has been rapidly growing and it now also includes prokaryotic glucose dehydrogenase, several hydroxysteroid dehydrogenases, and additional forms [7 -121. The recent finding that a mammalian 15-hydroxyprostaglandin dehydrogenase and other mammalian enzymes belong to this group [12-161, gives the family added interest also in eukaryotic metabolism. Little is known about the functional mechanisms for these short-chain enzymes, and we now show common properties and further members that establish both similarities
The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the Rl and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S.typhimurium nrdE and nrdF genes (RlE and R2F Ribonucleotide reductases catalyze the synthesis of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. At least three separate classes of enzymes are known, each with a distinct protein structure but all requiring a protein radical for catalysis (1). The long-studied aerobic Escherichia coli enzyme is the prototype for class I enzymes, also present in all higher organisms and some other microorganisms. E. coli genes nrdA and nrdB encode the a and 8 polypeptide chains, respectively, that form the Rl (a2) and R2 (132) proteins that constitute the enzyme (2). Each protomer of the Rl dimer (2 x 85.7 kDa) contains one substrate-binding site with redox-active thiols involved in the reduction of the substrate ribonucleoside diphosphate, and two separate types of allosteric sites: one, the activity site, controls the overall activity of the enzyme, with ATP as a positive effector and dATP as a negative effector; the other, the substrate-specificity site, controls the specificity of the enzyme, with ATP and dATP favoring pyrimidine reduction, dTTP favoring GDP reduction, and dGTP favoring ADP reduction (3).The R2 dimer (2 x 43.4 kDa) contains two dinuclear iron centers with associated stable tyrosyl free radicals, located at Tyr-122 of the polypeptide chain (4). The drug hydroxyurea scavenges this radical and thereby inactivates the enzyme. Class II and III enzymes lack the tyrosyl radical. Class II enzymes, with the Lactobacillus leichmanni enzyme as a prototype, employ adenosylcobalamin as a radical generator, whereas class III enzymes use S-adenosylmethionine together with iron for this purpose.Salmonella typhimurium contains an active class I enzyme with amino acid sequences 96.5% and 98.4% identical to the E. coli Rl and R2 proteins, respectively (A.J., unpublished results). Recent genetic evidence involving complementation of nrd mutants of E. coli suggested the presence in S. typhimurium of the genes, nrdE and nrdF, coding for a second class I enzyme (5). These genes are also present on the chromosome of E. coli but are, under standard growth conditions, expressed only when present on a plasmid. The amino acid sequences deduced for the corresponding proteins showed a limited identity with other class I enzymes but contained many of their catalytically important residues.We have purified and characterized the two proteins encoded by the cloned genes nrdE and nrdF from S. typhimurium. Each protein is a homodimer. Together they catalyze the reductio...
During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication. The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG. The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase. The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence, RVSGY, of pyruvate formate-lyase. During activation of the anaerobically induced pyruvate formate-lyase,
A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia cofi. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dAl, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map ofE. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dAl is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dAl activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.During anaerobic growth, Escherichia coli induces an enzyme that catalyzes the reduction of CTP to dCTP (7-9, 11). The gene for this enzyme was recently cloned (28) and found to be distinct from nrdA and nrdB, which code for the aerobic ribonucleoside diphosphate reductase (29). In the active state, both enzymes contain organic radicals as part of their protein structures and iron as a cofactor (19,29). In the aerobic enzyme, the radical is located on and the enzyme contains a diferric center with the iron ions linked by a ,u-oxo-bridge (29). A glycyl residue was suggested to harbor the organic radical of the anaerobic reductase, whose iron center consists of an iron-sulfur cluster (19,28).A difference between the two enzymes is that the aerobic reductase, as isolated, shows full activity and contains the radical in stable form, whereas the isolated anaerobic enzyme is inactive and lacks the radical. Instead, the enzyme activity and radical of the latter enzyme appear only after anaerobic incubation of the isolated protein with NADPH, S-adenosylmethionine, and two uncharacterized E. coli fractions, provisionally called dAl and RT (8
The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a Agtll library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map ofE. coil. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.
Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2e ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several dermed protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein Bi) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.Ribonucleotide reductases catalyze the synthesis of the four deoxyribonucleoside triphosphates (dNTPs) required for DNA replication (1-4). The enzymes provide a link between RNA and DNA metabolism. They have attained special biological interest in connection with recent theories concerning the evolution of life on earth since their appearance was a prerequisite for the transition of an "RNA world" into a "DNA world" (5). Several forms of the enzyme occur in nature, with different structures and catalytic mechanisms. Also, their chemistry is of considerable interest. The enzyme from Escherichia coli provided the first example of a biological process that uses a protein radical as a mechanistic option (6, 7). While the direct reduction of ribonucleotides via a radical mechanism is a general mode of dNPT synthesis in all organisms, the way in which this is achieved varies. At present, three classes of ribonucleotide reductases have been described: The aerobic E. coli enzyme (8) is the prototype for class I enzymes (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), also present in all higher animal (9) and plant cells (10). These enzymes consist of two proteins: one large homodimer (protein B1 of E. coli) and one small homodimer (protein B2). Characteristic features are the presence of a ferric:tyrosyl radical center in B2 (6) and redox-active dithiols in B1 (11), both involved in the catalytic process. B1 also provides the reductase with allosteric pro...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.