ABSTRACT
The gene encoding edeine B1 amidinohydrolase from Bacillus brevis TT02-8 was cloned into Escherichia coli and its nucleotide sequence was determined. An open reading frame was identified and was found to encode a polypeptide of 289 amino acid residues with a predicted molecular weight of 32,455, which was consistent with that previously calculated for edeine B1 amidinohydrolase purified from this bacterium. Comparison of the deduced amino acid sequence of this enzyme with other amidinohydrolases revealed the highest homology to B. subtilis agmatine ureohydolase. The enzymatic activity of the protein produced in Escherichia coli was analyzed. Three histidine residues, H-112, H-137 and H-151 in the edeine B1 amidinohydrolase, which are highly conserved in amidinohydrolases, were changed to alanine by site-directed mutagenesis. Analysis of each of these mutants revealed that three histidine residues are important but not essential for the enzyme activity.
Subject(s)
Bacillus/genetics , Escherichia coli/genetics , Ureohydrolases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Sequence Homology, Amino AcidSubject(s)
Anti-Bacterial Agents/metabolism , Bacillus/enzymology , Edeine/analogs & derivatives , Spermidine/analogs & derivatives , Ureohydrolases/chemistry , Amino Acid Sequence , Ammonium Sulfate/chemistry , Anti-Bacterial Agents/chemistry , Arginase/chemistry , Arginine/metabolism , Chemical Precipitation , Chromatography, Gel , Chromatography, High Pressure Liquid , Edeine/chemistry , Edeine/metabolism , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Molecular Sequence Data , Sequence Analysis , Spermidine/chemistry , Spermidine/metabolism , Substrate Specificity , Ureohydrolases/genetics , Ureohydrolases/isolation & purificationSubject(s)
Biosensing Techniques , Chaetomium/metabolism , Fatty Acids/chemistry , Proteins/chemistry , Receptors, Cell Surface , Animals , Arachidonic Acid/chemistry , Brain/metabolism , Carrier Proteins/genetics , DNA, Complementary/metabolism , Leptin , Linoleic Acid/chemistry , Magnetic Resonance Spectroscopy , Oleic Acid/chemistry , Palmitic Acid/chemistry , Proteins/drug effects , Proteins/metabolism , Rats , Receptors, Leptin , Spectrometry, Mass, Fast Atom Bombardment , Stearic Acids/chemistryABSTRACT
The gene from Bacillus brevis TT02-8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02-8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33-66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.
Subject(s)
Arginase/biosynthesis , Arginase/genetics , Bacillus/genetics , Bacillus/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genes, Bacterial/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Culture Media , DNA, Bacterial/biosynthesis , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Gene Library , Humans , Molecular Sequence Data , Open Reading Frames/genetics , Plasmids , Polymerase Chain Reaction , Rats , Species SpecificitySubject(s)
Anti-Bacterial Agents/pharmacology , Eimeria tenella/drug effects , Soil Microbiology , Streptomyces/metabolism , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Ethers/chemistry , Ethers/isolation & purification , Ethers/pharmacology , Fermentation , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microbial Sensitivity Tests , Spectrophotometry, InfraredABSTRACT
Blasticidin S (BS) deaminase (BSR) from a BS-resistant strain, Bacillus cereus K55-S1, was purified to homogeneity. Molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration on HPLC are about 15500 and 35000, respectively, indicating the enzyme is a homodimer. The amino acid composition and N-terminal sequence of BSR are the same as those deduced from the nucleotide sequence of the BS-resistant gene, bsr. The optimum temperature and pH for enzyme activity are 60-65 degrees C and near 10.0, respectively. The activity of BSR is inhibited by Cu2+, Hg2+, and p-chloromercuric benzoate (PCMB). Inhibition by PCMB or HgCl2 is reversible by the addition of SH reagents. The enzyme catalyzes the deamination of BS and its derivatives, but not cytosine nucleosides.
Subject(s)
Aminohydrolases/isolation & purification , Anti-Bacterial Agents/antagonists & inhibitors , Bacillus cereus/enzymology , Aminohydrolases/antagonists & inhibitors , Aminohydrolases/genetics , Aminohydrolases/metabolism , Bacillus cereus/genetics , Bacillus cereus/physiology , Chromatography, Liquid , Drug Resistance, Microbial , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Nucleosides/antagonists & inhibitors , Plasmids , Substrate Specificity , TemperatureABSTRACT
A bacterial arginase was purified to homogeneity from a strain of Bacillus brevis. The native enzyme, with an estimated MW of 143,000, migrated on SDS-PAGE as a single polypeptide of estimated MW of 33,000. The enzyme, highly specific to L-arginine, showed the maximum activity at pH 11.0 in the presence of Mn2+ ions and the pI was 4.8 by isoelectric focusing. The enzyme activity was increased significantly by the addition of Mn2+, Ni2+, or Co2+ ions, and inhibited potently by chemicals such as HgCl2, N-bromosuccinimide, or glutathione. The Kms for L-arginine and L-canavanine were 0.69 and 22.2 mM, respectively. The enzyme was inhibited competitively by gamma-guanidinobutyric acid, and non-competitively by L-lysine, L-ornithine, creatine, blasticidin S, and edeine B1. Analysis of the N-terminal amino acid sequence of the purified bacterial enzyme found 33-36% homologies with the Agrobacterium, yeast, rat, and human enzymes.
Subject(s)
Arginase/isolation & purification , Arginase/metabolism , Bacillus/enzymology , Amino Acid Sequence , Arginase/chemistry , Cations , Chromatography, DEAE-Cellulose , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Substrate Specificity , ThermodynamicsSubject(s)
Aminoglycosides , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/isolation & purification , Antibiotics, Antineoplastic/isolation & purification , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Leukemia P388/drug therapy , Melanoma, Experimental/drug therapy , MiceABSTRACT
To study the relationship between citrate synthase and 2-phosphinomethylmalic acid (PMM) synthase, which catalyzes a very similar reaction comparable to citrate formation in the biosynthesis of a herbicide, bialaphos, citrate synthase was purified from the mycelium of Streptomyces hygroscopicus SF-1293, a bialaphos-producing organism. The overall purification was 440-fold with a yield of 4.4% from cell-free extract. Based on comparison with PMM synthase, it has been concluded that citrate synthase of S. hygroscopicus is quite different from PMM synthase in several aspects such as enzymatic properties, amino acid composition. N-terminal amino acid sequence, and stereo-chemical reaction mechanism.
Subject(s)
Citrate (si)-Synthase/metabolism , Oxo-Acid-Lyases/metabolism , Streptomyces/enzymology , Amino Acid Sequence , Base Sequence , Citrate (si)-Synthase/genetics , Citrate (si)-Synthase/isolation & purification , Cloning, Molecular , DNA, Bacterial/genetics , Kinetics , Molecular Sequence Data , Oxo-Acid-Lyases/genetics , Restriction Mapping , Streptomyces/geneticsABSTRACT
2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.