Designing Thermostable Proteins: Ancestral Mutants of 3-Isopropylmalate Dehydrogenase Designed by using a Phylogenetic Tree (original) (raw)
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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.
Scientific Reports
Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from The...
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...
"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.
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.
Protein Science, 1998
We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Themus themophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a ZeuB-deficient strain of Z themophilus. The resulting transformant showed a leucine-autotrophy at 56 "C but not at 61 "C and above. Phenotypically thermostabilized strains that can grow at 61 "C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 "C and mutants that showed a leucine-autotrophic growth at 70 "C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.