Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8 (original) (raw)
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We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76؇C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-1723Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.
Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.
The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus
Gene, 1998
3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli. The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron-sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved.
"Protein Engineering, Design and Selection", 1995
Chimeric isopropylmalate dehydrogenases were constructed by connecting the genes isolated from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis. These genes were expressed in Escherichia coli. The enzymes were purified and analysed. Enzymes of TJhermophilus and Bsubtilis and chimeric enzymes showed similar enzymological characteristics except for thermal stability. The stability of each enzyme was approximately proportional to the content of the amino acid sequence from the TJhermophilus enzyme. The results suggested that amino acid residues contributing the thermal stability distribute themselves, in general, evenly at least in the Nterminal half of the amino acid sequence of TJhermophilus isopropylmalate dehydrogenase.
Screening of stable proteins in an extreme thermophile, Thermus thermophilus
Molecular Microbiology, 1995
The leuB gene codes for 3-lsopropylmalate dehydrogenase of the leucjne biosynthetic pathway in an extreme thermophile. Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucJne was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.
Extremophiles, 2007
Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T m , of 82.2 and 84.5°C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened Nterminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70°C. The pH-activity profile was bellshaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a threefold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T m of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60°C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK a s of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.
European Journal of Biochemistry, 1994
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 7: thermophilus enzyme is conferred by increased hydrophobic interaction at the subunit-subunit interface. Two residues at the interface of the 7: thermophilus enzyme, Leu246 and Va1249, 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 7: thermophilus enzyme [L246E, V249MlIPMDH 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.
European journal of biochemistry / FEBS, 1993
The gene for a L(+)-lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. Idh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine-Dalgarno sequence within the non-coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme. Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequence with those of known prokaryotic L-lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found. The gene was expressed as an active enzyme in a heterologous host.
Thermostability of ancestral mutants ofCaldococcus noboribetusisocitrate dehydrogenase
Fems Microbiology Letters, 2005
We constructed mutant genes of Caldococcus noboribetus isocitrate dehydrogenase containing ancestral amino acid residues that were inferred using the maximal likelihood method and a composite phylogenetic tree of isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase. The mutant genes were expressed in Escherichia coli and the protein products purified. Thermostabilities, reported as the half-inactivation temperatures, for the purified enzymes were determined and compared with that of the wild-type enzyme. Four of the five mutant enzymes have greater thermal stabilities than wild-type isocitrate dehydrogenase. The results are compatible with the hyperthermophilic universal ancestor (commonote) hypothesis. Incorporation of ancestral residues into a modern-day protein sequence can be used to improve protein thermostability.
Isocitrate dehydrogenase of the thermoacidophilic archaebacterium Sulpholobus acidocaldarius
FEBS Letters, 1984
The thermoacidophilic archaebacterium, Sulpholobus acidocaldarius, has been found to possess both NAD-and NADP-dependent isocitrate dehydrogenase activities. Evidence is presented to suggest that both enzymic activities are functions of the same protein: NAD' and NADP+ compete with each other for the enzyme and do so with K, values equal to their Km values: thermal inactivation results in the toss of both activities at the same rate and copurification was observed on gel filtration, ion-exchange chromatography and polyacrylamide gel electrophoresis. The evolutionary significance of this unique isocitrate dehydrogenase is discussed.