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Sulfur amino acid metabolism in Saccharomyces cerevisiae is regulated by the level of intracellular S-adenosylmethionine (AdoMet). Two cis-acting elements have been previously identified within the 5' upstream regions of the structural genes of the sulfur network. The first contains the CACGTG motif and is the target of the transcription activation complex Cbflp-Met4p-Met28p. We report here the identification of two new factors, Met31p and Met32p, that recognize the second cis-acting element. Met31p was isolated through the use of the one-hybrid method, while Met32p was identified during the analysis of the yeast methionine transport system. Met31p and Met32p are highly related zinc finger-containing proteins. Both LexA-Met31p and LexA-Met32p fusion proteins activate the transcription of a LexAop-containing promoter in a Met4p-dependent manner. Northern blot analyses of cells that do not express either Met31p and/or Met32p suggest that the function of the two proteins during the transcriptional regulation of the sulfur network varies from one gene to the other. While the expression of both the MET3 and MET14 genes was shown to strictly depend upon the presence of either Met31p or Met32p, the transcription of the MET25 gene is constitutive in cells lacking both Met31p and Met32p. These results therefore emphasise the diversity of the mechanisms allowing regulation of the expression of the methionine biosynthetic genes.
Mol Cell Biol 1997 Jul
PMID:Met31p and Met32p, two related zinc finger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism. 919 98

The S-adenosylhomocysteine (SAH) technique allows the estimation of the free cytosolic adenosine concentration using the kinetic properties of the enzyme SAH-hydrolase (adenosine+homocysteine reversible SAH+H2O). Besides the cytosolic adenosine concentration, the local SAH signal may also depend on the local homocysteine availability, the continuous production of SAH from S-adenosylmethionine (SAM-->SAH+CH3) and the activity of the enzyme SAH-hydrolase. These variables were studied with high spatial resolution (sample dry mass 25 mg) in left ventricular myocardium from 26 anesthetized open-chest dogs in which heart rate averaged 86 +/- 14 beats/min and mean aortic pressure 96 +/- 17 mmHg. Homocysteine infusion (48 mg/kg i.v.) increased the normal plasma homocysteine concentration from 5.0 +/- 0.8 to 586 +/- 40 microM after 30 min when the average tissue concentration was 94% of the plasma concentration and similar in low and high flow areas (flow range 0.04 to 1.91 ml/min/g). Local SAH content was 1.18 +/- 0.48 nmol/g under control conditions and increased to 4.33 +/- 0.59 nmol/g within 60 min following competitive blockage of the SAH-hydrolase by adenosine dialdehyde (10 mumol/kg i.v.). This increase of the SAH content was slightly more in high than in low-flow areas (P < 0.01). Regional SAH-hydrolase activity (9.0 +/- 0.5 nmol/min/g) was comparable in high and low flow areas. All three variables exhibited an observed variability which was larger than the methodical variability suggesting significant spatial heterogeneity in the myocardium. A regrouping analysis indicated that between four and five samples taken from distant sites should be averaged to obtain a robust estimate of the above metabolic parameters. Reconciling the measurements with a mathematical model of cardiac adenosine metabolism and fitting of the measured SAH tissue levels gave an estimate of 72 pmol/min/g for the mean transmethylation rate. Estimates of the cytosolic adenosine concentration of cardiomyocytes and endothelial cells under control physiological conditions were 24 and 7 microM, respectively. Thus, the present measurements provide a basis for the quantitative assessment of the local cytosolic adenosine concentration in relation to blood flow.
J Mol Cell Cardiol 1997 May
PMID:Determinants of the S-adenosylhomocysteine (SAH) technique for the local assessment of cardiac free cytosolic adenosine. 920 16

Pisatin is the major phytoalexin produced by pea upon microbial infection. The enzyme that catalyzes the terminal step in the pisatin biosynthetic pathway is (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM). We report here the isolation and characterization of two HMM cDNA clones (pHMM1 and pHMM2) made from RNA obtained from Nectria haematococca-infected pea tissue. The two clones were confirmed to encode HMM activity by heterologous expression in Escherichia coli. The substrate specificity of the methyltransferases in E. coli was similar to the activity detected in CuCl2-treated pea tissue. Nucleotide sequence analysis of Hmm1 and Hmm2 revealed an open reading frame of 1080 bp and 360 amino acid residues which would encode 40.36 kda and 40.41 kDa polypeptides, respectively. The deduced amino acid sequence of HMM1 has 95.8% identity to HMM2, 40.6% identity to Zrp4, a putative O-methyltransferase (OMT) in maize root, and 39.1% to pBH72-F1, a putative OMT induced in barley by fungal pathogens or UV light. Comparison of the deduced amino acid sequences of the cDNA clones to OMTs from other higher plants identified the binding sites of S-adenosylmethionine (AdoMet). Southern blot analysis showed two closely linked genes with strong homology to Hmm in the pea genome.
Plant Mol Biol 1997 Nov
PMID:Isolation of the cDNAs encoding (+)6a-hydroxymaackiain 3-O-methyltransferase, the terminal step for the synthesis of the phytoalexin pisatin in Pisum sativum. 934 77

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is coordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at < or =1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.
Mol Microbiol 1997 Oct
PMID:Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. 938 51

Various analogues of adenosine have been described as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, and some of these AdoHcy hydrolase inhibitors (e.g., 3-deazaadenosine, 3-deazaaristeromycin, and 3-deazaneplanocin A) have also been reported to inhibit the replication of human immunodeficiency virus type 1 (HIV-1). When evaluated against HIV-1 replication in MT-4 cells, macrophages, or phytohemagglutinin-stimulated peripheral blood lymphocytes infected acutely or chronically with HIV-1IIIB or HIVBaL strains, a wide range of adenosine analogues did not inhibit HIV-1IIIB replication for 50% at subtoxic concentrations. However, they inhibited HIV-1 replication in HeLa CD4+ LTR-LacZ cells at concentrations well below cytotoxicity threshold. A close correlation was found among the inhibitory effect of the compounds on AdoHcy hydrolase activity, their inhibition of HIV-1 replication in Hela CD4+ LTR-LacZ cells, and their inhibition of the HIV-1 Tat-dependent and -independent transactivation of the long terminal repeat, whereas no inhibitory effect was seen on HIV-1 reverse transcription or a Tat-independent cytomegalovirus promoter. Our results suggest that AdoHcy hydrolase and the associated S-adenosylmethionine-dependent methylation mechanism play a role in the process of long terminal repeat transactivation and, hence, HIV replication.
Mol Pharmacol 1997 Dec
PMID:S-adenosylhomocysteine hydrolase inhibitors interfere with the replication of human immunodeficiency virus type 1 through inhibition of the LTR transactivation. 939 86

It has been known for several decades that cyclopropane fatty acids (CFAs) occur in the phospholipids of many species of bacteria. CFAs are formed by the addition of a methylene group, derived from the methyl group of S-adenosylmethionine, across the carbon-carbon double bond of unsaturated fatty acids (UFAs). The C1 transfer does not involve free fatty acids or intermediates of phospholipid biosynthesis but, rather, mature phospholipid molecules already incorporated into membrane bilayers. Furthermore, CFAs are typically produced at the onset of the stationary phase in bacterial cultures. CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is noteworthy in several respects. It is catalyzed by a soluble enzyme, although one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. The enzyme, CFA synthase, discriminates between phospholipid vesicles containing only saturated fatty acids and those containing UFAs; it exhibits no affinity for vesicles of the former composition. These and other properties imply that topologically novel protein-lipid interactions occur in the biosynthesis of CFAs. The timing and extent of the UFA-to-CFA conversion in batch cultures and the widespread distribution of CFA synthesis among bacteria would seem to suggest an important physiological role for this phenomenon, yet its rationale remains unclear despite experimental tests of a variety of hypotheses. Manipulation of the CFA synthase of Escherichia coli by genetic methods has nevertheless provided valuable insight into the physiology of CFA formation. It has identified the CFA synthase gene as one of several rpoS-regulated genes of E. coli and has provided for the construction of strains in which proposed cellular functions of CFAs can be properly evaluated. Cloning and manipulation of the CFA synthase structural gene have also enabled this novel but extremely unstable enzyme to be purified and analyzed in molecular terms and have led to the identification of mechanistically related enzymes in clinically important bacterial pathogens.
Microbiol Mol Biol Rev 1997 Dec
PMID:Cyclopropane ring formation in membrane lipids of bacteria. 940 47

Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.
Microbiol Mol Biol Rev 1997 Dec
PMID:Metabolism of sulfur amino acids in Saccharomyces cerevisiae. 940 50

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
Mol Microbiol 1998 Apr
PMID:In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone. 959 7

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5' of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic beta-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.
Mol Gen Genet 1998 Apr
PMID:Analysis of eryBI, eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea. 961 75

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.
Biochem Mol Biol Int 1998 Jun
PMID:DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64. 963 38


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