The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase (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.
Protein Engineering Design and Selection, 2002
Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (∆T 1 2 ⍧ 0-13°C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k cat and K m values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k cat /K m values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in freeenergy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (∆H ‡) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k cat value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126.
FEBS Letters, 1997
To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3-isopropylmalate dehydrogenase in which Ala 172 was replaced with Asp, Glu, Asn, and Gin were prepared by site-directed mutagenesis. Three mutants were more stable than the wild-type enzyme. No significant change in catalytic properties was found in the mutant enzymes. The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core.
Journal of Molecular Biology, 2006
We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.
Journal of bacteriology, 1996
A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the genera...
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.
"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.
Biochemistry, 2016
The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the k cat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (K m values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1−13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate K m values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme−substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure. 3-Isopropylmalate dehydrogenase (IPMDH) is a member of the β-hydroxyacid oxidative decarboxylase family, to which also isocitrate dehydrogenase, homoisocitrate dehydrogenase, tartarate dehydrogenase, and malic enzyme belong (cf. ref 1 for a review). Evidence from structural, 2−4 bioinformatics, 1 and biochemical studies (mutation analysis, pH profiles, etc.) 4−8 suggests that the catalytic apparatus of these related enzymes are similar (including the critical role of a Lys-Tyr pair), although there may be some uncertainties concerning the role of each amino acid side chain in the active site. In particular, 49 contradictory conclusions were drawn about the contribution of 50 the active site aspartates in the catalysis by isocitrate 51 dehydrogenase. As for IPMDH, functional studies based on 52 mutational analysis are rather scarce, 10−12 and the role of the 53 active site Lys and Asp sid-chains has not yet been tested.
Crystal structures of mutants of Thermus thermophilus IPMDH adapted to low temperatures
Protein Engineering Design and Selection
Random mutagenesis on thermophilic 3-isopropylmalate dehydrogenases (IPMDH; EC 1.1.1.85) produced mutant enzymes which adapt to low temperatures. These mutants had higher activity at lower temperatures than the wild-type enzyme without losing high thermostability. Here we report three structures of ... [more]