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PubMed2002

The disulfide redox system of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase.

Schistosoma mansoni, a causative agent of schistosomiasis, is a major cause of human morbidity in tropical countries. Adult schistosomes, which reside in the hepatic portal system, are exposed to reactive oxygen compounds through respiration and as a result of the host immune response. To minimize oxidative stress schistosomes must possess adequate mechanisms of detoxification. Major detoxification systems rely on reducing equivalents from the disulfide oxidoreductases glutathione and thioredoxin. Therefore, maintenance of adequate levels of these thiols in a reduced form is critical. Here we show that S. mansoni possess an unusual thiol redox system centered on thioredoxin glutathione reductase. This enzyme represents an unusual fusion of a pyridine nucleotide disulfide oxidoreductase with a redox active glutaredoxin extension. Furthermore, we predict that this is a selenocysteine protein. Immunoprecipitation, western blot and inhibitor studies show that this protein has thioredoxin reductase, glutathione reductase, and glutaredoxin activities. Most importantly, we show that thioredoxin glutathione reductase appears to be the major, if not the sole enzyme for these activities in adult worms, completely replacing thioredoxin reductase and glutathione reductase. This is the first example of an organism with a redox system based exclusively on thioredoxin glutathione reductase.

Amino Acid Sequence↗
PubMed1985

Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae.

The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity. During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.

Aminohydrolases↗
PubMed1986

Biosynthesis of cyclosporin A: partial purification and properties of a multifunctional enzyme from Tolypocladium inflatum.

An enzyme fraction most probably involved in the biosynthesis of cyclosporin A was purified 540-fold from Tolypocladium inflatum. The enzyme was capable of forming covalent enzyme-substrate complexes and catalyzed the ATP-pyrophosphate exchange reactions dependent on the unmethylated constituent amino acids of cyclosporin A. Evidence was obtained that covalent binding of substrate amino acids occurred via thioester linkage. Furthermore, the N-methylation of thio-esterified valine, leucine, and glycine residues with S-adenosyl-L-methionine was demonstrated. De novo synthesis of cyclosporin A was not observed but the formation of the diketopiperazine cyclo-(D-Ala-MeLeu) from D-alanine and L-leucine under the consumption of ATP and S-adenosyl-L-methionine. This cyclodipeptide represents a partial sequence of cyclosporin A. Molecular mass determinations revealed the enzyme activity to be lying in the range of about 700 kDa.

Carbon Radioisotopes↗
PubMed1989

Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme.

C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Structure-based mutational and functional analysis identify human NM23-H2 as a multifunctional enzyme.

The human NM23-H2 protein is a transcriptional regulator (PuF) that binds and cleaves DNA via covalent bond formation, and also catalyzes phosphoryl transfer (NDP kinase). Our previous work has identified two separate DNA-binding regions on NM23-H2/PuF: a sequence-dependent DNA-binding surface involving residues Arg34, Asn69, and Lys134 on the equator of the hexameric protein and a covalent DNA-binding site involving Lys12 located in the nucleotide-binding site, the site of the NDP kinase reaction. To understand the role of the nucleotide-binding site in the DNA cleavage reaction and to establish a connection between the nuclease and the NDP kinase activities, we used the known crystal structure of NM23-H2 complexed with GDP as the basis for site-directed mutagenesis. We thus identified Arg88 and Arg105 as residues that are, in addition to Lys12, critical for covalent DNA binding and DNA cleavage, as well as for the NDP kinase reaction. Another residue, Gln17, was required only for DNA cleavage, and Tyr52, Asn115, and His118 were found to be essential only for the NDP kinase activity. Six of these seven functionally important amino acids associated with the nucleotide-binding site are evolutionarily conserved, underscoring their biological importance. We also show that nucleoside triphosphates but not nucleoside diphosphates inhibited the covalent DNA binding and DNA cleavage reactions, independent of phosphoryl transfer and the NDP kinase reaction. These findings collectively suggest that the binding modes of mononucleotides and duplex DNA oligonucleotides in the nucleotide-binding site differ, and that NM23-H2 possesses multiple biochemical activities. A model consistent with these observations is presented.

Adenosine Diphosphate↗
PubMed2006

Cysteine cathepsins: multifunctional enzymes in cancer.

Cysteine cathepsins are highly upregulated in a wide variety of cancers by mechanisms ranging from gene amplification to post-transcriptional modification. Their localization within intracellular lysosomes often changes during neoplastic progression, resulting in secretion of both inactive and active forms and association with binding partners on the tumour cell surface. Secreted, cell-surface and intracellular cysteine cathepsins function in proteolytic pathways that increase neoplastic progression. Direct proof for causal roles in tumour growth, migration, invasion, angiogenesis and metastasis has been shown by downregulating or ablating the expression of individual cysteine cathepsins in tumour cells and in transgenic mouse models of human cancer.

Cysteine Endopeptidases↗
PubMed1996

Long-term alcohol intake enhances ADP-ribosylation of the multifunctional enzyme, phosphoglucomutase, in rat liver.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational protein modification that, in turn, alters several regulatory proteins in mammalian cells. We demonstrated that long-term alcohol intake enhanced the ADP-ribosylation of a 58-kd protein in rat liver plasma membranes. To assess the biological significance of this phenomenon, we partially purified the 58-kd acceptor protein from solubilized rat liver homogenates by two sequential preparative high-pressure liquid chromatographies. Microsequencing revealed that it was phosphoglucomutase (PGM) (EC 5,4,2,2). This enzyme underwent negligible auto ADP-ribosylation, but the ADP-ribosylation was remarkably increased by adding rat liver plasma membranes. The extent of the increase was greater in alcohol-fed rats than in pair-fed controls, suggesting enhanced enzyme activities toward ADP-ribosylation of PGM after chronic alcohol consumption. Several important enzymes are ADP-ribosylated, after which their activities are modified. The results of this study showed that PGM is a novel substrate for ADP-ribosylation in the liver and that the ADP-ribosylation is increased after chronic alcohol consumption. In view of the variety of roles of PGM in the liver (carbohydrate metabolism and Ca2+ homeostasis), specific roles of this modification in terms of the effects of alcohol on hepatocytes may deserve further investigation.

Adenosine Diphosphate Ribose↗
PubMed1989

Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases.

Lipoprotein lipase is an important regulator of lipid and lipoprotein metabolism. It also contributes to the lipid and energy metabolism of different tissues in varying ways. Although the synthesis, manner of secretion, and mechanism of endothelial binding of lipoprotein lipase appear similar in all tissues, the factors that control gene expression and posttranslational events related to processing vary from tissue to tissue. The actual molecular events that determine this tissue specificity are not yet understood. In the future, however, it may be possible to stimulate or inhibit the activity of lipoprotein lipase in specific tissues and to alter metabolic processes so as to improve the quality and length of life in patients with metabolic diseases such as hypertriglyceridemia, HDL2 deficiency, and obesity.

Humans↗
PubMed1977

phiX174 cistron A protein is a multifunctional enzyme in DNA replication.

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.

Coliphages↗
PubMed1994

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of clavaminate synthase from Streptomyces antibioticus. A multifunctional enzyme of clavam biosynthesis.

Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.

Amino Acid Sequence↗