Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii
Michika Sasaki
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Mayumi Uno
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Satoshi Akanuma
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Akihiko Yamagishi
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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)