Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^– mutants were selected in vivo after transformation of the modified plasmid into a ura3 yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to 70 × 10^–4, i.e. 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed 48% frameshifts, 44% base substitutions and 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5-(A/T)_nG-3 where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

Purification and properties of N 5 ,N 10 -methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogens known so far. N ⁵ N ¹⁰ -Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO₂, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F₄₂₀ as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V _max at 65°C and pH 6.8 was 435 U/mg (k_cat=275 s^-1) and the K _m for methylenetetrahydro-methanopterin and for reduced F₄₂₀ were 6 M and 4 M, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q ₁₀ between 40°C and 90°C was 1.5.The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity.The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance.The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵ ,N ¹⁰ -methylene-H₄MPT - CH₃-H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT⁺ N ⁵ ,N ¹⁰ -methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀; 1 U=1 mol/min     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

Purification and characterization of homodimeric methylmalonyl-CoA mutase from<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

High activity (>60 munit/mg protein) of 5-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000±5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0±2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH₂-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.Abbreviations AdoCbl 5-Deoxyadenosylcobalamin - KPB Potassium phosphate buffer - MCM Methylmalonyl-CoA mutase - PVDF Polyvinylidene difluoride     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Purification and structural properties of adenylylated and deadenylylated glutamine synthetase from Rhodopseudomonas sphaeroides

Glutamine synthetase (GS) of Rhodopseudomonas sphaeroides is regulated by adenylylation and deadenylylation. The extent of adenylylation/deadenylylation of the enzyme in cell free extracts was influenced by inorganic phosphate (P _i), -ketoglutarate, ATP and other nucleotides. While P _i and -ketoglutarate stimulated deadenylylation, ATP and other nucleotides enhanced adenylylation of the GS. By using proper combinations of the effectors and incubation conditions, any desired adenylylation state of GS could be adjusted in vitro. The enzyme was purified to electrophoretic homogenity by three steps including affinity chromatography on 5-AMP-Sepharose. Adenylylated and deadenylylated enzyme showed different UV-spectra and isoelectric points. The native enzyme had a molecular weight of 600,000, deadenylylated subunits of 50,000±1,000. Electron microscopic investigations revealed a dodecameric arrangement of subunits in two hexameric planes.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose     

Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli

Summary The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl--d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent M _r of approximately 61000. The calculated M _r of the ced1 gene product was 61023.Abbreviations Ap ampicillin - ced1 gene coding for Ced1 - Ced1 cellodextrinase from B. fibrisolvens - CMC carboxymethylcellulose - LB Luria Bertani - ORF open reading frame - pNPC p-nitrophenyl--d-cellobioside - PC phosphate citrate - HCA hydrophobic cluster analysis     

Co-occurrence of aflatoxin B<Subscript>1</Subscript> and cyclopiazonic acid in sour lime (<Emphasis Type="Italic">Citrus aurantifolia Swingle</Emphasis>) during post-harvest pathogenesis by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B₁ producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 g/kg) than aflatoxin B₁ (ranging from 141.3 to 811.7 g/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B₁ and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.     

Purification and characterization of β-xylosidase from <Emphasis Type="Italic">Streptomyces</Emphasis>sp. CH7 and its gene sequence analysis

A thermostable -xylosidase was extracted and purified from Streptomyces sp. CH7 mycelium. The apparent molecular weight of the native enzyme estimated by gel filtration was around 173 and 87 kDa for the two subunits estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its optimal pH and temperature were 6.5 and 55 °C, respectively. The enzyme was stable at pH 6–9 and at 50 °C after 30 min. The K _m values for p-nitrophenyl---xylopyranoside and o-nitrophenyl---xylopyranoside were 0.56 and 0.94 mM with the V _m values of 26.3 and 6.6 U/mg protein, respectively. The enzyme was inhibited strongly by Hg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺. It was inhibited by xylose competitively for p-nitrophenyl---xylopyranoside with the K _i value of 40 mM. Characterization of the nucleotide sequence of pCH7-1 carrying the -xylosidase gene from Streptomyces sp. CH7 revealed 3 open reading frames (ORF). The first truncated ORF, bxlI, encodes a putative ABC-type sugar transport system, permease component. The second ORF, bxl2, encodes -xylosidase, while the third truncated ORF, bxl3, encodes a putative oxidoreductase. The deduced 791 amino acid sequence of Bxl2 showed 84, 71 and 66% identity to those of Streptomyces coelicolor, Streptomyces lividansand Streptomyces thermoviolaceus, respectively. The calculated molecular mass of the deduced amino acid sequence revealed close similarity to that of the purified enzyme.     

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of - and -subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K_m=0.77 M) than for 2-aminophenol (K_m=0.89 M) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the - and -subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the - and -subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/K M) and thermostability in the presence of salts

The activity of purified N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K _m for N ⁵,N ¹⁰-methenyltetrahydromethanopterin from 2 mM to 40 M and by increasing the V _max from 2000 U/mg (k_cat=1385 s^-1) to 13300 U/mg (k_cat=9200 s^-1). Besides increasing the catalytic efficiency (k_cat/K _m) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.Abbreviations H₄MPT tetrahydromethanopterin - N ⁵,N ¹⁰-methenyl-H₄MPT - CHO-H₄MPT N ⁵-formyl-H₄-MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄-MPT N ⁵-methyl-H₄MPT - MOPS -N-morpholinopropane sulfonic acid - TRICINE N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Pectinmethylesterase isoforms fromVigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform

Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE was determined. The nature protein is composed of 318 amino acids with a calculatedM _r of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in theV _max and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.     

A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an _{2 2} oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg²⁺ was required for activity. The pH optimum was 8 at 1 mM PP _i and 5 mM Mg²⁺. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent K _m for PP _i of 0.1 mM in the presence of 5 mM Mg²⁺. The V _max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K _cat of 1400 per second.The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedyre were applied.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum

An inducible sulfite reductase was purified from Clostridium pasteurianum. The pH optimum of the enzyme is 7.5 in phosphate buffer. The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: ₂₂. Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm. No siroheme chromophore was isolated from this reductase. The enzyme could reduced the following substrates in preferential order: NH₂OH> SeO ₃ ^2- >NO ₂ ^2- at rates 50% or less of its preferred substrate SO ₃ ^2- . The proposed dissimilatory intermediates, S₃O ₆ ^2- or S₂O ₃ ^2- , were not utilized by this reductase while KCN inhibited its activity. Varying the substrate concentration [SO ₃ ^2- ] from 1 to 2.5 mol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H₂ uptake to S^2- formed from 2.5:1 to 3.1:1. The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen. Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.Abbreviations SDS sodium dodecyl sulfate - BSA bovine serum albumin - LDH Lactate dehydrogenase