1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium.
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1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression. (+info)
The crystal structures of mutated 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 and their relationship to the thermostability of the enzyme.
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The structures of two mutant forms (G240A and L246E/V249M) of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 were studied by X-ray crystallography. In the case of G240A, the replacement of glycine by alanine at residue 240 was expected to decrease the thermostability as a result of abnormal contacts between the methyl group of alanine and the peptide chain. However, the normal van der Waals' contacts were achieved owing to a shift in a bundle of beta-strands that yielded a vacant space for the alanine residue. The extended hydrogen bonds within the beta-sheet are the major reason for the decreased thermostability of G240A. The mutations in L246E/V249M are located in an alpha-helix region which is involved in subunit-subunit contact via hydrophobic interaction. Loosening of the subunit-subunit contact owing to ionic repulsion was the major cause of the lower heat stability of L246E/V249M. (+info)
Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase.
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The Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two functionally and evolutionarily related enzymes with distinct substrate specificities. To understand the determinants of substrate specificities of the two proteins, the substrate and coenzyme in IPMDH were docked into their respective binding sites based on the published structure for apo IPMDH and its sequence and structural homology to ICDH. This modeling study suggests that (1) the substrate and coenzyme (NAD) binding modes of IPMDH are significantly different from those of ICDH, (2) the interactions between the substrates and coenzymes help explain the differences in substrate specificities of IPMDH and ICDH, and (3) binding of the substrate and coenzyme should induce a conformational change in the structure of IPMDH. (+info)
Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis.
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Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes. The leuA complementing fragment encoding the isopropylmalate synthase was sequenced. The leuA gene is 1,848 bp in size, encoding a polypeptide with an M(r) of 68,187. Upstream of leuA there is extensive hyphenated dyad symmetry and a putative leader peptide, which are features characteristic of attenuation control. In addition to leuA, the sequenced fragment contains an open reading frame with high coding probability whose disruption did not result in a detectable phenotype. Furthermore, the sequence revealed that this open reading frame separates leuA from lysC, which encodes the aspartate kinase initiating the synthesis of all amino acids of the aspartate family. The leuA gene was inactivated in three lysine-secreting strains by insertional mutagenesis. Fermentations were performed, and a roughly 50% higher lysine yield was obtained when appropriate leucine concentrations limiting for growth of the constructed strains were used. (+info)
Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus.
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We cloned and sequenced the leuB gene encoding 3-isopropylmalate dehydrogenase from Escherichia coli K-12 (JM103). Errors (33 residues) were found and corrected in the sequence previously reported for the leuB gene of Thermus thermophilus. The three-dimensional structure of the thermophile enzyme and the amino acid sequence comparison suggested that a part of the high stability of the T. thermophilus enzyme is conferred by increased hydrophobic interaction at the subunit-subunit interface. Two residues at the interface of the T. thermophilus enzyme, Leu246 and Val249, are substituted with less hydrophobic residues, Glu and Met, respectively, in the E. coli enzyme, whereas other residues in this region are highly conserved. The mutated T. thermophilus enzyme [L246E, V249M]IPMDH had reduced stability to heat. Two residues of the E. coli dehydrogenase, Glu256 and Met259, were replaced with the corresponding residues from the thermophile sequence. The resulted mutant enzyme was more resistant to heat than the wild-type enzyme. (+info)
Overexpression of genes of an extreme thermophile Thermus thermophilus, in Escherichia coli cells.
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The 3-isopropylmalate dehydrogenase gene from an extreme thermophile, Thermus thermophilus, was not expressed in Escherichia coli unless a palindromic structure around the ribosome binding site was eliminated or a leader open reading frame was introduced into the upstream flanking region of the gene. This report suggests a way to increase the expression of this gene, with a high G+C content, in E. coli. (+info)
3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization.
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3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasmid containing the leuB gene. The native enzyme molecule is a dimer of molecular weight 38,000. The Km value for 3-isopropylmalate was estimated to be 26 microM and that for NAD+ 0.8 mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0 mM Mg2+ or Co2+. The optimum pH and temperature for the activity are 9.0 and 60 degrees C, respectively. The properties of the enzyme are similar to those of the Salmonella typhimurium and Thermus thermophilus enzymes, except for substrate specificity. T. ferrooxidans 3-isopropylmalate dehydrogenase is able to utilize D- and L-malate as substrates in addition to 3-isopropylmalate. Sequencing of subcloned DNA revealed that the leuB gene consists of a 1,074 bp open reading frame and encodes 358 amino acid residues corresponding to the subunit (38,462 Da). The amino acid sequence of 3-isopropylmalate dehydrogenase from T. ferrooxidans and those of some heterotrophic microorganisms have high homology. (+info)
Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona.
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The leuB gene of Leptospira interrogans serovar pomona strain kenniwicki has been cloned on a 9.5 kb plasmid, pWVL1, by complementation of Escherichia coli leuB mutants. Subcloning and Tn5 mutagenesis showed that the region required for complementation was approximately 1.2 kb in length. Enzyme assays showed that the product of the cloned gene was a beta-isopropylmalate dehydrogenase. Defects in the leuA, leuC and leuD genes of E. coli were not complemented by pWVL1. The nucleotide sequence of the leuB-complementing region and surrounding DNA has been determined. Three open reading frames were found which encode proteins of 40.9, 38.8 and 15 kDa. Analysis of subclones containing nucleotide deletions of varying sizes showed that only the 38.8 kDa protein was necessary to obtain complementation of E. coli leuB mutations. The PIR data base was searched and the enzyme 3-isopropylmalate dehydrogenase from six different micro-organisms was found to share significant amino acid sequence similarity (43-57%) with the 38.8 kDa L. interrogans leuB gene product. The organization of the leucine biosynthetic genes in L. interrogans differs from that found in E. coli, Salmonella typhimurium and Bacillus subtilis. (+info)