Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

Protein Engineering Design and Selection, Dec 2008

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the Km values for NAD (nicotinamide adenine dinucleotide) and/or the kcat values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.

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Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

Michika Sasaki 0 Mayumi Uno 0 Satoshi Akanuma 0 Akihiko Yamagishi 0 0 Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences , 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan 1To whom correspondence should be addressed. E-mail: In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the Km values for NAD (nicotinamide adenine dinucleotide) and/ or the kcat values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme. Introduction An increasing number of thermostable enzymes are being isolated from thermophilic organisms. In addition to their thermostabilities, these enzymes are also unusually resistant to the effects of other protein-inactivating agents, such as organic solvents, acidic and alkaline pHs, and detergents (Suzuki et al., 2001). Their extreme stabilities make thermophilic enzymes attractive tools for industrial processes (Vieille et al., 1996; Haki and Rakshit, 2003; van den Burg, 2003). However, a crucial limitation to the design of such industrial processes is that thermostable enzymes are nearly inactive at more moderate temperatures temperatures at which their less stable mesophilic or psychrophilic counterparts have maximum activities. Recently, efforts have been made to improve the lowtemperature catalytic activities of thermophilic enzymes. Mutations that improve the catalytic activity of Pyrococcus furiosus b-glucosidase CelB at low temperatures have been found by Lebbink et al. (2000). Merz et al. (2000) selected Sulfolobus solfataricus (S. solfataricus) indoleglycerolphosphate isomerase mutants that are catalytically more active at 378C than is the wild-type enzyme. Low-temperature adaptation of the thermophilic Thermus thermophilus (T. thermophilus) xylose isomerases enzymatic activity has also been reported (Lo nn et al., 2002). Random mutagenesis of a rationally designed, low-temperature adapted mutant of the extremely thermostable Thermotoga neapolitana xylose isomerase produced additional mutants with improved lowtemperature adaptations (Sriprapundh et al., 2003). While these examples clearly demonstrate that one or a small number of mutations can improve low-temperature activity, predictive rules, based on physical and/or chemical guidelines, have not yet been established. 3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85), the product of the leuB gene, is an enzyme involved in leucine biosynthesis. The leuB gene has been cloned from a variety of micro-organisms including the extreme thermophile, T. thermophilus HB8 (Tanaka et al., 1981) and the hyperthermophile Sulfolobus tokodaii (S. tokodaii) (Suzuki et al., 1997). The catalytic properties, the thermal stability, and the tertiary structure of the unusually thermostable T. thermophilus IPMDH are well characterized (Yamada et al., 1990; Imada et al., 1991). Some T. thermophilus IPMDH mutants that are catalytically more active than the wild-type enzyme at temperatures ranging from 30 to 408C have been isolated from a library composed of randomly mutated leuB genes (Suzuki et al., 2001; Yasugi et al., 2001). Detailed analyses of the kinetic properties of these low-temperature adapted mutants show that the effects caused by their mutations are of two types. Some substitutions contribute to an increased kcat value; whereas, others only result in an improved Km value for the coenzyme NAD (nicotinamide adenine dinucleotide). To date, no T. thermophilus IPMDH mutant exists that has both improved kcat and Km values. For the study reported herein, we focused on the most thermostable IPMDH characterized to date, which is that of S. tokodaii. Its biophysical properties and tertiary structure are known (...truncated)


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Michika Sasaki, Mayumi Uno, Satoshi Akanuma, Akihiko Yamagishi. Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii, Protein Engineering Design and Selection, 2008, pp. 721-727, 21/12, DOI: 10.1093/protein/gzn054