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PubMed1989

Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis.

There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP). In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway. Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 +/- 0.3 kilobases. Expression of gene ura2 is regulated at the transcriptional level. As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame. The deducted amino acid sequence has allowed the identification of four domains. Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase. There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains. The protein has been purified as a multienzyme aggregate and as a multifunctional protein. The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons. Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro. ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus. Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells. The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate. Dihydroorotate dehydrogenase activity was found in the cytoplasm. Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10. The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate. The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA.

Analysis of the primary structure of peptide synthetases involved in non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure. These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation. To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His6-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATP-dependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA-His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His). However, it retained the functions of specific amino acid activation and thioester formation. The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP-dependent activation as well as in thioester formation and racemization.

Adenosine Triphosphate↗
PubMedDate not supplied

[Thrombin: a multifunctional enzyme].

Thrombin is the final enzyme of blood coagulation cascade. It belongs to the trypsin family of serine proteases. Its two primary actions are to cleave fibrinogen to release fibrin and to activate platelets through a limited proteolysis of a specific receptor. In addition, thrombin is the major regulator of blood coagulation. It is both a procoagulant enzyme in the activation of factors V and VIII, and an anticoagulant enzyme through the activation of protein C and TAFI. This multi-functionality of thrombin depends upon the conformation of its active site: depth for high specificity and shape for a finely tuned selection of substrates. Since new anticoagulant molecules, some with anti-thrombin activity, are emerging, it is important to understand the mechanisms allowing thrombin to be so specifically multifunctional.

Anticoagulants↗
PubMed2001

The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis.

Recent studies have shown that the genes of the gibberellin (GA) biosynthesis pathway in the fungus Gibberella fujikuroi are organized in a cluster of at least seven genes. P450-1 is one of four cytochrome P450 monooxygenase genes in this cluster. Disruption of the P450-1 gene in the GA-producing wild-type strain IMI 58289 led to total loss of GA production. Analysis of the P450-1-disrupted mutants indicated that GA biosynthesis was blocked immediately after ent-kaurenoic acid. The function of the P450-1 gene product was investigated further by inserting the gene into mutants of G. fujikuroi that lack the entire GA gene cluster; the gene was highly expressed under GA production conditions in the absence of the other GA-biosynthesis genes. Cultures of transformants containing P450-1 converted ent-[(14)C]kaurenoic acid efficiently into [(14)C]GA(14), indicating that P450-1 catalyzes four sequential steps in the GA-biosynthetic pathway: 7beta-hydroxylation, contraction of ring B by oxidation at C-6, 3beta-hydroxylation, and oxidation at C-7. The GA precursors ent-7alpha-hydroxy[(14)C]kaurenoic acid, [(14)C]GA(12)-aldehyde, and [(14)C]GA(12) were also converted to [(14)C]GA(14). In addition, there is an indication that P450-1 may also be involved in the formation of the kaurenolides and fujenoic acids, which are by-products of GA biosynthesis in G. fujikuroi. Thus, P450-1 displays remarkable multifunctionality and may be responsible for the formation of 12 products.

Base Sequence↗
PubMed1996

[Lipoprotein lipase: a multifunctional enzyme in lipoprotein metabolism].

Lipoprotein lipase (LPL) is a rate-limiting enzyme for the hydrolysis of triglycerides. Recently new insights into non-enzymatic functions have emerged. Complete lipoprotein lipase deficiency associated with chylomicronemia is an uncommon (1/10(6) in the general population) autosomal recessive disorder caused by many different lipoprotein lipase gene mutations and is characterized by high fasting plasma triglyceride levels, that can be complicated with acute pancreatitis. To date, about sixty gene mutations have been described throughout the world. Conversely to the homozygous state, the heterozygous state predisposes to a lipid profile that may be atherogenic evenly frequent (approximately 1/500) in the general population. These new clinical and biological insights reinforce the multifunctional role of lipoprotein lipase.

Acute Disease↗
PubMed1994

Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase.

Enniatin synthetase catalyzes the biosynthesis of N-methylated cyclohexadepsipeptides. The 347 kDa enzyme is encoded by the esyn1 gene of Fusarium scirpi and contains two domains (EA and EB) homologous to each other and to regions of other microbial peptide synthetases. Parts of the esyn1 gene were subcloned in frame to a small lacZ gene portion of Escherichia coli expression vectors. Overproduced recombinant proteins showed a high tendency towards inclusion body formation and could be only partially dissolved in 8 M urea or 6 M guanidine hydrochloride. After renaturation, a 121 kDa recombinant protein representing the N-terminal conserved domain EA of enniatin synthetase was shown to activate D-hydroxyisolvaleric acid via adenylation. Similarly, a 158 kDa recombinant protein comprising the C-terminal conserved domain EB catalyzed the activation of the substrate amino acid (e.g. L-valine). Moreover, this protein could be photolabeled with S-[methyl-14C]adenosyl-L-methionine, (AdoMet) indicating the presence of the methyltransferase. Both functions, L-valine activation and AdoMet binding, could be assigned to a 108 kDa recombinant protein encompassing the A and the M segment of domain EB. The fact that a 65 kDa recombinant protein representing the M portion could be photolabeled, indicated the localization of the methyltransferase in this region. Three deletion mutants of the 65 kDa protein were shown to be inactive with respect to UV-induced AdoMet labeling.

Amino Acid Sequence↗
PubMed1998

Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex.

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.

Amino Acid Sequence↗
PubMed1999

Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?

Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

Thermodynamics of the reactions catalyzed by the multifunctional enzyme complex tryptophan synthase.

The intrinsic enthalpy changes (corrected for hydration of D-glyceraldehyde 3-phosphate) for the reactions catalyzed by the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli have been determined calorimetrically. Cleavage of indoleglycerol phosphate (alpha reaction) was found to be associated with a delta H value of 54.0 +/- 2.5 kJ mol-1, while condensation of indole with L-serine (beta reaction) involved -80.3 +/- 4.6 kJ mol-1'. By direct determination of the enthalpy concomitant with the overall synthesis of tryptophan from indoleglycerol phosphate and L-serine an enthalpy value of -13.4 +/- 5.6 kJ mol-1 was observed. In view of the uncertainties of the literature data used for calculation of the hydration contribution, the agreement between the directly measured delta H value of the overall reaction and the sum of the enthalpies of the alpha and beta reactions is fair. Deamination of L-serine, a side reaction catalyzed preferentially by the isolated beta 2 pyridoxal 5'-phosphate2 subunit, was shown to be associated with an enthalpy change of -7.3 +/- 0.4 kJ mol-1.

Buffers↗
PubMed1978

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5) one of the activities of a trifunctional folate-dependent enzyme from porcine liver, uses an ordered kinetic mechannism as determined from initial velocity, product inhibition and dead-end inhibition studies. The final product released from the dehydrogenase is methenyltetrahydrofolate. However, from the time course of appearance of products it is observed that the methenyltetrahydrofolate, rather than equilibrating with the solution, is converted preferentially to formyltetrahydrofolate by the cyclohydrolase, (5,10-methenyletrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9) demonstrating a functional interaction between these two enzymic activites.

Aminohydrolases↗
PubMed1991

6-Hydroxymellein synthetase as a multifunctional enzyme complex in elicitor-treated carrot root extract.

Synthetic activity of a polyketide compound 6-hydroxymellein was induced in elicitor-treated carrot root tissues. The activity was significantly inhibited by an antiserum raised against the acyl carrier protein (ACP) of fatty acid synthetase, suggesting that the enzyme(s) for 6-hydroxymellein synthesis require(s) a functional unit similar to ACP. However, the synthetic activity was not stimulated by the addition of ACP purified from Escherichia coli and was not lost even after fractionation by gel-filtration chromatography. The active fraction obtained by gel-filtration (136 kDa) was subjected to immunoblot analysis, and a 128 kDa polypeptide in the fraction was found to cross-react with anti-ACP serum. These observations suggest that the biosynthesis of 6-hydroxymellein in carrot cells is catalyzed by an enzyme consisting of a single peptide chain.

Acyl Carrier Protein↗
PubMed1987

A eukaryotic repressor protein, the qa-1S gene product of Neurospora crassa, is homologous to part of the arom multifunctional enzyme.

Little is known about the proteins involved in the control of gene expression in eukaryotes. Although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. We report here that the qa-1S repressor protein of the fungus Neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukaryotes. We believe that this is the first known case of a repressor protein that is unequivocally homologous to metabolic enzymes. These findings allow us to propose likely functions for some regions of the qa-1S protein.

Amino Acid Sequence↗
PubMed1990

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme.

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.

Chromatography, Gel↗
PubMed1995

Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography.

Cyclic AMP affinity chromatography applied to various mammalian tissue extracts yielded two proteins in addition to the regulatory subunits of protein kinase. This paper characterizes these proteins and provides a simple procedure for their preparation. The polypeptides (36 kDa and a 19 kDa/21 kDa doublet) were isolated from the cAMP matrix by sequential elution with cAMP solutions of increasing concentrations. Microsequencing was accomplished following chemical or enzymic degradation of isolated polypeptides. Partial amino acid sequences of the 36 kDa protein and analyses of its enzymic activity indicated identity with glyceraldehyde-3-phosphate dehydrogenase whilst the lower MW protein proved to be identical with mammalian nucleoside diphosphate kinase subunits. In both cases, binding to cAMP appeared to occur at the nucleotide (NAD and ATP, respectively) sites. In conclusion, we present a one step-procedure, applicable to tissue and cell extracts, which allows the simultaneous isolation of both glyceraldehyde-3-phosphate dehydrogenase and nucleoside diphosphate kinase. This procedure may help to elucidate the multiple functions of these two important enzymes.

Amino Acid Sequence↗