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PubMed1995

Two domains with amino-acid sequence similarity are required for dihydroneopterin aldolase function in the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii.

The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) codes for a multifunctional enzyme (Fas) known to catalyse three consecutive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway. Previously, we predicted that a domain, designated FasB (amino acids (aa) 161-280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathway, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa identity with the DHNA of Streptococcus pneumoniae (Sp). We now extend these findings to show that a second domain, FasA (aa 39-160), whose function was previously unknown, shares 27% sequence identity with the adjacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were constructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect cells. No DHNA activity is associated with either fasA or fasB when produced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced FasAB polypeptide. The results of aa sequence alignments and expression studies suggest that FasA and FasB may be two subunits of the DHNA enzyme moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed.

Aldehyde-Lyases↗

PubMed2005

Relationship between enzyme activity and dimeric structure of recombinant HIV-1 reverse transcriptase.

The multifunctional enzyme human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer composed of a 66-kDa (p66) subunit and a p66-derived 51-kDa (p51) subunit. p66/p51 HIV-1 RT contains 1 functional DNA polymerase and 1 ribonuclease H (RNase H) active site, which both reside in the p66 subunit at spatially distinct regions. In this study, we have investigated the relationship between the heterodimeric structure of HIV-1 RT and its enzymatic properties by introducing mutations at RT codon W401 that inhibit the formation of p66/p51 heterodimers. We demonstrate a striking correlation between abrogation of both HIV-1 RT dimerization and DNA polymerase activity. In contrast, the p66 monomers exhibited only moderately slowed catalytic rates of DNA polymerase-dependent and DNA polymerase-independent RNase H cleavage activity compared with the wild-type (WT) enzyme. Furthermore, no major changes in the unique cleavage patterns were observed between the WT and mutant enzymes for the different substrates used in the RNase H cleavage assays. Based on these results, and on our current understanding of HIV-1 RT structure, we propose that the p66 monomer can adopt an open tertiary conformation that is similar to that observed for the subunit in the heterodimeric enzyme. We also propose that the formation of intersubunit interactions in HIV-1 RT regulates the establishment of a functional DNA polymerase active site.

Binding Sites↗

PubMed1996

Kinamycin acetyltransferase I from Streptomyces murayamaensis, an apparently large, membrane-associated enzyme.

Identification and initial characterization of an apparently large, membrane-associated multifunctional enzyme, kinamycin acetyltransferase I (KAT I), is described. KAT I activity was enriched 29-fold over the level in cell-free extracts of Streptomyces murayamaensis. Two acetyltransferase activities catalyzing acetyl coenzyme A dependent conversion of kinamycin F and E to kinamycin E and D, respectively, were inseparable in the course of the partial purification. Partial purification involved separation of KAT I from cytosolic proteins by differential ultracentrifugation, solubilization with 0.5% CHAPS zwitterionic detergent followed by ultracentrifugation, and Sephacryl S400 gel filtration chromatography of the resulting supernatant.

Acetyltransferases↗

PubMed1998

Engineering of solvent-exposed loops in Escherichia coli beta-galactosidase.

The Escherichia coli beta-galactosidase is a high molecular mass tetrameric enzyme extensively used as a molecular marker. Despite its proven utility as a partner in fusion proteins, previous attempts to generate insertional mutants rendered inactive or poorly active enzymes, hampering its further engineering for the construction of multifunctional enzymes. We have explored several solvent-exposed loops on the tetramer, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segments on the surface of active beta-galactosidase enzymes. An RGD-containing antigenic peptide positioned in these sites interacts efficiently with specific monoclonal antibodies as well as target integrins on the surface of mammalian cells. The resulting chimeric enzymes are soluble, stable, produced in high yields and enzymatically active. Moreover, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors.

Amino Acid Sequence↗

PubMed1985

Limits of adaptation: the evolution of selective neutrality.

Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form. The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness. Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number. At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral. We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection. Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli. Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways. We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations. The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.

Adaptation, Physiological↗

PubMed1994

Interleukin-2 transcriptional block by multifunctional Ca2+/calmodulin kinase.

In the presence of costimulation, Ca2+ influx in T cells leads to activation (transcription of interleukin-2; ref. 2) via calcineurin. In the absence of costimulation, Ca2+ influx results in anergy (interleukin-2 transcriptional block) through an unknown mechanism. Specific attenuation of interleukin-2 transcriptional induction occurs in Jurkat T cells following pretreatment with a Ca2+ ionophore. A > 90% block of inducible interleukin-2 reporter gene activity was initiated by transfection of a constitutively active mutant of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase or CaM kinase II), but not by constitutive mutants of CaM kinase IV, calcineurin or protein kinase C. The block was complete six hours after kinase transfection and showed specificity for interleukin-2; there was no change in beta-actin transcription or in c-fos transcription induced by phorbol myristyl acetate, and a Rous sarcoma virus promoter was stimulated threefold. Multifunctional CaM kinase also attenuated interleukin-2 activation by calcineurin plus phorbol ester. T-cell receptor signalling activates multifunctional CaM kinase. These findings suggest that two Ca2+/calmodulin-responsive enzymes, multifunctional CaM kinase and calcineurin, could mediate the divergent effects of Ca2+ signals in T-lymphocyte regulation.

Amino Acid Sequence↗

PubMed2002

Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A.

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.

Acyltransferases↗

PubMed2005

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal.

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

Aldehydes↗

PubMed1999

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.

Bacterial Toxins↗

PubMed1988

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway.

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

Alcohol Oxidoreductases↗

PubMed1993

Type selective inhibition of microbial fatty acid synthases by thiolactomycin.

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.

Anti-Bacterial Agents↗

PubMed2001

Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center.

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.

Acyltransferases↗

PubMed1992

Multidomain enzymes involved in peptide synthesis.

Biosynthesis of peptides in non-ribosomal systems is catalyzed by multifunctional enzymes that employ the thio-template mechanism. Recent studies on the analysis of the primary structure of several peptide synthetases have revealed that they are organized in highly conserved and repeated functional domains. The aligned domains provide the template for peptide synthesis, and their order determines the sequence of the peptide product.

Amino Acid Sequence↗

PubMed2006

Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1.

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75-80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.

Alanine↗

PubMed1977

[Assembly and function of organized enzymes (author's transl)].

When different enzymes form complexes with one another they gain new capabilities of function. Tryptophan synthase is discussed as an illustrative example in which formation of the complex leads to mutual activation of the component subunits. Kinetic studies show that conformational changes play an important role in this process. As the extreme case of self-organisation, structural genes of different enzymes might have been fused in the course of evolution, giving rise to multifunctional enzymes.

Biological Evolution↗

PubMed1996

Transacylase-like structure and its role in substrate channeling of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cell extracts.

6-Hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, lost the binding ability toward its co-substrates, acetyl- and malonyl-CoAs, by the treatment with the blocking reagents for serine-OH. In contrast, the enzyme retained the binding ability even when the two SH groups at the reaction center (cysteine-SH of the condensation enzyme and cysteamine-SH of acyl carrier protein) were blocked, and one substrate bound to the SH-blocked enzyme was readily replaced by the other. It appeared that the cysteine-SH accepted only acetyl moiety while cysteamine-SH was preferentially malonylated in the presence of both of the substrates. These results suggest that transacylase-like domain is involved in the structure of 6-hydroxymellein synthase as a common primary binding site of its co-substrates, and acetyl and malonyl moieties are properly channeled from their CoA esters to cysteine-SH and acyl carrier protein-SH via this domain, respectively.

Acetyl Coenzyme A↗

PubMed2000

Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells.

6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation and the ketoreduction in the presence of the nucleotide reducing co-factors. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable inhibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of the NADH-associated enzyme while, in the presence of NADPH, the compound slightly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deuterium-labeled compound, however, the kH/kD value was markedly reduced if NADH and [D]NADH were employed as the reducing co-factors. These results suggest that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an important role in the regulation of the enzyme activity.

Journal Article↗

PubMed1996

Rabbit aorta glutathione S-transferases and their role in bioactivation of trinitroglycerin.

The pharmacological action of glyceryl trinitrate (GTN), a widely used drug for the treatment of angina pectoris, is thought to be mediated through release of nitric oxide (NO) during its biotransformation. Since glutathione S-transferases (GST) can utilize GTN as substrate and GST inhibitors can attenuate GTN-induced relaxation of rabbit aorta in vitro it has been suggested that these enzymes are involved in the bioactivation of GTN in rabbit aorta. Because GSTs are multifunctional enzymes and a multitude of GST isozymes with varying substrate preferences are present in mammalian tissues, the role of specific GST isozymes in bioactivation of GTN in rabbit aorta needs to be established. Therefore, during the present studies we have purified and characterized GST isozymes from rabbit aorta and evaluated their possible roles in the biotransformation of GTN. The results of these studies showed that rabbit aorta contained three GST isozymes having pI values of 9.4, 7.7, and 5.4. Structural, immunological, and kinetic studies showed that GST 9.4, GST 7.7, and GST 5.4 belonged to the alpha-, pi-, and mu-classes, respectively. The relative abundance of these enzymes in rabbit aorta was alpha > pi > mu. The alpha- and mu-class GST isozymes had similar activities toward GTN (0.71 U/mg and 0.86 U/mg, respectively) while the pi-class GST showed much lower activity toward GTN. The catalytic efficiency k(cat)/Km of the mu- and alpha-class GSTs toward GTN were similar but these activities were differentially inhibited by ethacrynic acid, its GSH conjugate, bromosulfophthalein (BSP), and hematin. These results suggest that in rabbit aorta GSTs may be involved in bioactivation of GTN, and because of their higher abundance the alpha-class GSTs may be more important for the pharmacological effects of GTN than the mu-class GSTs. The results on kinetics of inhibition by various inhibitors suggest that hematin may be an effective inhibitor to delineate the role of specific GST isozymes in the bioactivation of GTN.

Amino Acid Sequence↗