Crystal structures of mutants of Thermus thermophilus IPMDH adapted to low temperatures (original) (raw)
Related papers
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.
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.
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.
Patterns of adaptation in a laboratory evolved thermophilic enzyme
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
The heat sensitive psychrophilic protease subtilisin S41 was previously subjected to three rounds of mutagenesis/ recombination and screening, resulting in variant 3-2G7, whose half-life at 60³C is approx. 500 times that of wild-type. Here we report the results of five additional generations of laboratory evolution starting from 3-2G7. The half-life of 8th generation enzyme 8-4A9 at 60³C is 1200 times that of wild-type, and slightly more than twice that of 3-2G7. This half-life is s 20-fold greater than those of homologous mesophilic subtilisins SSII and BPNP. Circular dichroism melting curves indicate that subtilisin 8-4A9 unfolds at temperatures approx. 25³C higher than wild-type. It is also substantially more resistant to proteolysis at 30³C. Nearly half of the 13 amino acid substitutions accumulated in 8-4A9 involve the mutation of serine residues. This mirrors a pattern observed in natural proteins, where serines are statistically less prevalent in thermophilic enzymes compared to mesophilic ones. ß
Improvements of thermophilic enzymes: From genetic modifications to applications
Bioresource Technology, 2019
Thermozymes (from thermophiles or hyperthermophiles) offer obvious advantages due to their excellent thermostability, broad pH adaptation, and hydrolysis ability, resulting in diverse industrial applications including food, paper, and textile processing, biofuel production. However, natural thermozymes with low yield and poor adaptability severely hinder their large-scale applications. Extensive studies demonstrated that using genetic modifications such as directed evolution, semi-ration design, and rational design, expression regulations and chemical modifications effectively improved enzyme's yield, thermostability and catalytic efficiency. However, mechanism-based techniques for thermozymes improvements and applications need more attention. In this review, stabilizing mechanisms of thermozymes are summarized for thermozymes improvements, and these improved thermozymes eventually have large-scale industrial applications.
"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.
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.
Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis
European Journal of Biochemistry, 2002
Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wildtype and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEMÒ-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k cat ) for D-xylose compared with the wild-type enzyme at 60°C, but they did not show any increase in catalytic eciency (k cat /K m ). For D-glucose, both the k cat and the k cat /K m values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K i ) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular¯exibility of the mutant enzymes at low temperatures.
FEBS Letters, 2006
Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70°C when compared with the wild-type IPMDH.