ABSTRACT
Single crystals of citrate synthase from the Archaeon Thermoplasma acidophilum were obtained in two forms using the hanging drop vapour diffusion method and polyethylene glycol 3350 as precipitant. Type 1 crystals belong to the orthorhombic space group P222(1), with unit cell dimensions a = 80.9 A, b = 103.8 A, c = 98.3 A and one dimer in the asymmetric unit. Type 2 crystals belong to the monoclinic space group P2(1), with unit cell dimensions a = 53.8 A, b = 173.8 A, c = 86.7 A and beta = 97.1 degrees and two dimers in the asymmetric unit.
Subject(s)
Citrate (si)-Synthase/ultrastructure , Thermoplasma/enzymology , Citrate (si)-Synthase/isolation & purification , Crystallography , Protein Conformation , X-Ray DiffractionABSTRACT
The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17-residue oligonucleotide probes reverse translated from protein N-terminal sequence data. A 1670-bp BamH1-EcoR1 restriction fragment was ligated into pUC19 and pUC18 (constructs pTaGDH1 and pTaGDH2, respectively) and cloned in Escherichia coli. The sequence of the whole fragment was determined, and a 1059-bp open reading frame identified as the gene encoding glucose dehydrogenase. Cell-free extracts from E. coli carrying construct pTaGDH1 displayed glucose dehydrogenase activity indistinguishable from controls, but extracts from cells carrying pTaGDH2 displayed a 600-fold increase in glucose dehydrogenase activity. For high-level expression and purification of native protein, the glucose dehydrogenase coding sequence was subcloned into pMEX8. Glucose dehydrogenase purified from E. coli expressing the pMEX8 construct was indistinguishable by SDS/PAGE, N-terminal amino-acid sequence and kinetic analysis from the native enzyme purified from Tp. acidophilum. The derived 352-amino-acid sequence shows less than 20% identity with the glucose dehydrogenases of Bacillus subtilis and Bacillus megaterium but, by comparison with other eubacterial and eukaryotic dehydrogenase sequences, a portion of its sequence has been tentatively identified as a cofactor-binding region.
Subject(s)
Cloning, Molecular , Gene Expression , Glucose Dehydrogenases/chemistry , Glucose Dehydrogenases/genetics , Thermoplasma/enzymology , Amino Acid Sequence , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Glucose 1-Dehydrogenase , Molecular Sequence Data , Protein Biosynthesis , Sequence Analysis , Sequence Analysis, DNA , Thermoplasma/genetics , Transformation, BacterialABSTRACT
The nucleotide sequence of a gene encoding an L-2-haloalkanoic acid halidohydrolase from Pseudomonas putida strain AJ1 was determined. The ORF (hadL) codes for a polypeptide of 227 amino acids (Mr 25,687) which has significant homology to two other L-2-haloalkanoic acid halidohydrolases of Pseudomonas sp., DehcI and DehcII; these show 38% and 51% amino acid identity respectively to HadL. All three enzymes produce products of an opposite optical configuration to that of the substrates. Comparison of the three sequences shows several highly conserved motifs which indicate the possible position of the enzyme active site. The enzyme was purified to homogeneity and appears to exist as a tetramer.
Subject(s)
Bacterial Proteins/isolation & purification , Genes, Bacterial , Hydrolases/genetics , Pseudomonas putida/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Chromosome Mapping , Enzyme Activation , Hydrogen-Ion Concentration , Hydrolases/chemistry , Hydrolases/isolation & purification , Molecular Sequence Data , Pseudomonas putida/enzymologySubject(s)
Citrate (si)-Synthase/genetics , Citrate (si)-Synthase/isolation & purification , Genes, Bacterial , Halobacteriaceae/enzymology , Amino Acid Sequence , Base Sequence , Chromatography , Chromatography, Gel , Chromatography, Ion Exchange , Citrate (si)-Synthase/metabolism , Cloning, Molecular , Durapatite , Halobacteriaceae/genetics , Hydroxyapatites , Kinetics , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesisABSTRACT
Single crystals of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group P6122 or P6522, with unit cell dimensions a = b = 121.9 angstrom, c = 229.6 angstrom and with two molecules in the asymmetric unit.
Subject(s)
Bacterial Proteins/ultrastructure , Glucose Dehydrogenases/ultrastructure , Thermoplasma/enzymology , Crystallography , Glucose 1-Dehydrogenase , Protein Conformation , X-Ray DiffractionABSTRACT
A method for the routine profiling of illicit heroin samples received in casework has been developed which depends on simple and straightforward sample pretreatment, followed by gas chromatography on a capillary column using flame-ionization detection. The factors affecting the choice of each aspect of the procedure are discussed, as are the statistical data for sampling and the chromatography. Components of illicit heroin derived from opium and other adulterants have been identified. The significance of data from samples examined in 1986 is discussed.
Subject(s)
Forensic Medicine , Heroin/analysis , Chromatography, Gas , Drug ContaminationABSTRACT
The glutamate dehydrogenase gene of Escherichia coli has been cloned into broad host-range plasmids and can complement glutamate synthase mutants of Methylophilus methylotrophus. Assimilation of ammonia via glutamate dehydrogenase is more energy-efficient than via glutamate synthase, thus the recombinant organism converts more growth substrate, methanol, into cellular carbon.
