Structural analysis of 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 and the nonpiezophile Shewanella oneidensis MR-1.
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Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8.
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Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid. The thermostable enzyme thus produced was extracted from the E. coli cells, purified, and crystallized. The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x 10(4). The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M and that for NAD 6.3 x 10(-4) M. The optimum pH at 75 degrees C in the presence of 1.2 M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl. The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein. Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed. (+info)
Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.
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The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site. (+info)
Crystallization and preliminary X-ray data for 3-isopropylmalate dehydrogenase of Thermus thermophilus.
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The gene coding for 3-isopropylmalate dehydrogenase of Thermus thermophilus was cloned and expressed in Escherichia coli. The extracted enzyme was crystallized in a suitable size for X-ray crystallographic studies. The crystals have a space group of P3(1)21 or P3(2)21 with a = b = 78.6 A and c = 157.4 A. (+info)
Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis.
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A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76.2% in nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene. (+info)
Cloning of leucine genes of alkalophilic Bacillus No. 221 in E. coli and B. subtilis.
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The leucine genes of alkalophilic Bacillus No. 221 were cloned into the HindIII sites of pBR322 and pGR71, and recombinant plasmids pHK101 and pHK111 were constructed. The cloned 7.2 kb DNA consisting of HindIII fragments of 4.0 kb and 3.2 kb could substitute for leucine genes (alpha-isopropylmalate (alpha-IPM) synthetase gene, beta-IPM dehydrogenase gene, and alpha-IPM isomerase gene) of E. coli and B. subtilis, but not ilvB gene of B. subtilis. The 4.0 kb HindIII fragment could substitute for alpha-IPM synthetase gene of E. coli, and the 3.2 kb HindIII fragment could substitute for alpha-IPM isomerase gene of E. coli. Both 4.0 kb and 3.2 kb fragments are necessary to substitute for beta-IPM dehydrogenase gene. The expression of beta-IPM dehydrogenase gene of alkalophilic Bacillus No. 221 was repressed by the addition of leucine in the culture medium of B. subtilis carrying the plasmid pHK111. These results indicated that the 7.2 kb fragment contains the leucine gene cluster and its regulatory region. (+info)
The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number.
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The copy number of 2 mu DNA-derived plasmids in CIR+ Saccharomyces cerevisiae transformants is determined by its selective marker and is usually much lower than that of the endogenous plasmid. Only plasmids containing the leu2 allele of pJDB219, designated as leu2-d, under selective conditions displayed a higher copy number than did endogenous 2 mu DNA and by displacement generated cured cells. Spontaneous loss of 2 mu DNA occurred with a frequency of about 0.02% per generation. Curing plasmids, like pMP78, have copy numbers of 35; noncuring plasmids, like pDB248 or YEp6, have copy numbers of 4 to 8. The 2 mu DNA copy number in strains AH22 and YNN27 were determined to be 40 and 100, respectively. The high copy number of leu2-d-containing plasmids can be explained by its weak expression of less than 5% that of the wild-type LEU2 gene. The leu2-d allele has a deletion of the 5'-end sequence starting from 29 base pairs before the ATG initiation codon, but surprisingly, its expression is still regulated. On YRp7, which contains the chromosomal autonomic replication sequence ARS1, the defective leu2-d allele could not complement a leu2 host strain. This suggests a more stringent control of replication of ARS1-containing plasmids than of 2 mu-containing plasmids. (+info)