1 Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, 2 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 14321, Hachioji, Tokyo 192-0392 and 4 Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 111, Tsukuba 305-8572, Japan
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Abstract |
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Keywords: cold-adaptation/3-isopropylmalate dehydrogenase/random mutagenesis/thermal stability/thermophilic enzyme
Abbreviations: IPM, 3-isopropylmalate IPMDH, 3-isopropylmalate dehydrogenase (EC 1.1.1.85, IPMDH) MNNG, N-methyl-N'-nitronitrosoguanidine
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Introduction |
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Random mutagenesis has been utilized to enhance or alter various enzyme characteristics, including enzyme activity (Graham et al., 1993; Krebs and Fierke, 1993
; Huang et al., 1996
; Vipond and Halford, 1996
), thermal stability (Liao et al., 1986
; Tamakoshi et al., 1995
; Giver et al., 1998
), alkaline stability (Commingham and Wells, 1987
) and substrate specificity (Oliphant and Struhl, 1989
; Yano et al., 1998
), and also to restore the catalytic activity of an enzyme damaged by site-directed mutagenesis (Hermes et al., 1990
; Giver et al., 1998
). Momose and co-workers have isolated several mutant enzymes of subtilisin with improved catalytic activity at low temperature (Kano et al., 1997
; Taguchi et al., 1998
, 1999
). Lebbink et al. (2000) have isolated mutant enzymes with improved low-temperature catalysis of Pyrococcus furiosus ß-glucosidase CelB. Merz et al. (2000) have isolated mutants with improved catalytic activity at low temperature of the trpC gene of Sulfolobus solfataricus. However, detailed analysis of the adaptation mechanism to lower temperature has not yet been reported.
In this paper, we describe the application of random mutagenesis and isolation of cold-adapted mutant enzymes of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus. IPMDH encoded by leuB gene catalyzes the oxidative decarboxylation of 3-isopropylmalate in the presence of NAD as a cofactor and is an ideal enzyme for the present study because many mesophilic, thermophilic and psychrophilic counterparts with different thermal stabilities and catalytic properties (Yamada et al., 1990; Kirino et al., 1994
; Wallon et al., 1997a
, b
; Zavodszky et al., 1998
) are known to have close structural similarities (Imada et al., 1991
; Hurley and Dean, 1994
; Kadono et al., 1995
). In this study, T.thermophilus leuB gene was introduced into a leuB locus of Escherichia coli genome by using a newly constructed integration vector and cold-adapted mutant enzymes with higher activity at 40°C were isolated by screening the random mutant library. The isolated mutant enzymes were then characterized to elucidate the adaptation mechanism of enzymes to lower temperatures.
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Materials and methods |
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E.coli OM17, a leuB-deficient strain of E.coli JM105, was used in this study. The medium used was either LB broth or M9 minimum medium (Sambrook et al., 1989). Ampicillin (50 µg/ml), chloramphenicol (30 µg/ml), kanamycin (5 µg/ml) and isopropyl-ß-D-thiogalactopyranoside (IPTG, 4 µg/ml) were added to the media when required. Plasmid pMAK705 with a temperature-sensitive replication origin was a kind gift from Dr C.M.Hamilton (Hamilton et al., 1989
).
Construction of an integration vector for leuB locus
A 4.4 kbp HindIIIBamHI fragment including E.coli leu'ABC' genes (Kirino et al., 1994) was cloned in a plasmid pUC19 and used as a starting material. Two recognition sites, NdeI and PstI, were introduced at the initiation and near the termination codons of the leuB gene, respectively, by the method of Kunkel (Kunkel and Roberts, 1987
) using two synthetic oligonucleotides (5'-CAATATGTAATTCTAGACCATATGTCAGGTTTCCTTGTTG-3',5'-ATTGCCCGCT-ATGCTGCAGAAGGGGTG-3'). The E.coli leuB gene bordered by the NdeI and PstI sites was removed from the resulted plasmid and an NdeIXbaI fragment encoding T.thermophilus leuB gene (Tamakoshi et al., 1995
) and an XbaIPstI fragment encoding kanamycin nucleotidyl transferase gene (Matsumura et al., 1984
) were inserted between NdeI and PstI sites of the plasmid. To terminate the transcription from an upstream promoter, a trpA transcription-terminator sequence (Sambrook et al., 1989
) was synthesized (5'-AGCTTGTGAGCCCGCCT-AATGAGCGGGCTTTTTTTT-3',5'-CGCGAAAAAAAAG-CCCGCTCATTAGGCGGGCTCACA-3') and inserted between HindIII and MluI sites of the fragment. The resulting 5.0 kbp HindIIIBamHI fragment was inserted into the plasmid pMAK705 (Hamilton et al., 1989
) previously digested by HindIII and BamHI. The resulting plasmid was named pTS-EKT6 (Figure 1
).
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Integration of the vector pTS-EKT6 was performed by the method of Hamilton (Hamilton et al., 1989) with some modifications (Figure 1
). E.coli OM17 harboring the plasmid pTS-EKT6 was cultivated on an LB agar plate containing chloramphenicol at 44°C to isolate the transformants in which the vector is integrated into the genome. The isolation was repeated three times to delete intact vectors completely from the cells. Then, the colonies isolated from the plates were cultivated on LB agar plates supplemented with kanamycin at 30°C to clip out the vector from the genome. The colonies were purified by repeated cultivation. Finally, the clipped-out vector in the cell was removed by incubation of the isolated transformants on LB agar containing kanamycin at 44°C. They were purified by repeated plating and culturing and one of the strains was named TS4.
Mutagenesis with MNNG and screening of cold-adapted mutant strains
Mutagenesis with a mutagen, MNNG, was performed according to the method described by Miller (1992). E.coli TS4 cells treated with MNNG were incubated on M9 minimum medium agar plates containing kanamycin at 40°C for 3 days. Colonies appeared on the agar plates were purified by repeated plating and culturing and used for the following analyses.
Sequence analyses
The leuB genes of the isolated strains were amplified by a polymerase chain reaction with the synthetic primers 5'-ACAACGAAAACAACAAGGAAACC-3' and 5'-GTCATTATTATTGGTCCATTCAC-3'. The amplified fragments were digested with NdeI and EcoRV and cloned into the plasmid pNV119 (Tamakoshi et al., 1995) previously digested with NdeI and EcoRV. The sequence of the cloned genes was determined by the dideoxy chain termination method (Sanger et al., 1977
).
Expression and purification of mutant IPMDHs
The E.coli OM17 harboring the plasmid with each cloned leuB gene was cultured in LB broth containing ampicillin and IPTG to overexpress the mutated IPMDH. Wild-type and the mutant enzymes were purified following the method described previously (Yamada et al., 1990). All the enzymes used in this study were purified to homogeneity as judged by SDSPAGE (data not shown).
Analytical procedures
The specific activity of IPMDH was measured in 50 mM HEPES buffer (pH 8.0) containing 100 mM KCl, 5.0 mM MgCl2, 2.0 mM NAD and 1.0 mM D-3-isopropylmalate (IPM). The initial velocity of the reaction was determined by spectrophotometrically monitoring the reduction of NAD at 340 nm. The concentration of NADH was calculated by using a molar extinction coefficient of 6330 M1 cm1. Michaelis constants and kcat values were obtained by fitting initial velocity data to the MichaelisMenten equation. Thermodynamic parameters, i.e. activation free energy (G
), enthalpy (
H
) and entropy (
S
) and van't Hoff free energy (
GKm), enthalpy (
HKm) and entropy (
SKm), were derived according to the relationships given by Lehrer and Barker (1970).
Analysis of the irreversible thermal inactivation was performed by the method described previously (Kirino et al., 1994). Enzyme solution (0.2 mg/ml) was incubated at the indicated temperature for 10 min and immediately chilled in icewater. Then, aggregated protein was removed by centrifugation (12 000 g for 20 min). Residual activity was determined by measuring the catalytic activity of the supernatant.
The thermal denaturation profile was recorded by measuring the circular dichroism at 222 nm with a scanning rate of 1°C/min using a Model-720 spectropolarimeter (JASCO). A quartz cuvette with a 1 mm pathlength was used for measuring the circular dichroism. Temperature was monitored by using a CuNi thermocouple inserted into the cuvette.
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Results and discussion |
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For the efficient screening of cold-adapted mutant enzymes, it is important to avoid isolation of mutants with undesirable mutations such as the mutations to induce increased copy number of the gene. Therefore, T.thermophilus leuB gene was introduced into a leuB locus of E.coli genome in this study, since the copy number of the thermophilic leuB gene introduced is expected to be stable in the host genome.
T.thermophilus leuB gene and kanamycin nucleotidyl transferase gene were introduced into a leuB locus of E.coli genome (Blattner et al., 1997) by using the integration vector pTS-KET6 (Figure 1
). The vector possessed the 5.0 kbp HindIIIBamHI fragment containing the E.coli 'leuA, T.thermophilus leuB, kanamycin nucleotidyl transferase (Kmr) and E.coli leuC' genes. The chloramphenicol acetyl transferase gene (Cmr) was used to select transformants harboring the vector. E.coli OM17 was transferred with the vector. The vector also has a temperature-sensitive replication origin (Hamilton et al., 1989
) that inactivates at 40°C and above, which is useful for removing the vector from the host cells. E.coli OM17 harboring the integration vector was cultivated on LB agar plates containing chloramphenicol at 44°C. At this temperature the vector cannot replicate because of the inactivation of the temperature-sensitive replication origin. Consequently, only the cells with the vector integrated into the genome are expected to survive. The integration vector pTS-EKT6 is designed to have sequences identical with E.coli genome at 'leuA and leuC' loci. Accordingly, the homologous recombination with E.coli genome was expected to occur in one of two regions, i.e. the 1.1 kbp MluINdeI and 1.5 kbp PstIBamHI regions (*1 and *2 in Figure 1
). Subsequently, the isolated strains were cultured on LB agar plates containing kanamycin at 30°C. The plasmid replicon on the genome is active at the temperature applied, which inhibits the original replication of the genome (Hamilton et al., 1989
). Hence only the strains without the integrated vector are expected to grow efficiently. There are four possible recombinations, indicated by *3, *4, *5 and *6 in Figure 1
. The transcription terminator located upstream of the fragment also enables us to select the strain in which Kmr and T.thermophilus leuB genes are integrated into the genome by using kanamycin. Only the recombination at position *4 or *5 can promote expression of Kmr gene and therefore the recombinants of the two types are expected to be selected on kanamycin plates at 30°C. Then, the isolated strains were cultured on a kanamycin plate at 44°C to remove the clipped-out vector from the cell. Thus, by using this vector system, the T.thermophilus leuB gene and kanamycin nucleotidyl transferase gene can be substituted for E.coli leuB gene on the bacterial genome without monitoring the enzyme activity of the introduced leuB gene. One of the strains obtained was named TS4 and used for the following screening experiments.
Screening of cold-adapted mutant IPMDHs
The E.coli TS4 showed cold sensitivity in the leucine-free medium. The strain could grow only at 42°C and above without leucine. The observed cold sensitivity can be ascribed to the low activity of the T.thermophilus IPMDH at moderate temperature (Zavodszky et al., 1998). The strains were treated with a mutagen, MNNG and screened on leucine-free M9 minimum medium plate containing kanamycin at 40°C.
About 100 colonies appeared on the plate within 3 days per 7.5x106 surviving cells. The apparent efficiency was 1.2x104. Five strains were randomly selected from the colonies and labeled TS4-P115, -P215, -P315, -P415 and -T1155, respectively. The T.thermophilus leuB gene of each mutant was amplified by PCR and cloned into a plasmid pNV119 (Tamakoshi et al., 1995). Sequence analyses of the cloned leuB genes showed 13 base-substitutions per gene, except for TS4-P315 of which leuB gene had no mutations (Table I
). Four of the six detected substitutions were G:C to A:T transitions, as expected from the effect of the mutagen MNNG (Miller, 1992
). Four of the mutant leuB genes were overexpressed in E.coli and the expressed mutant enzymes, P115, P215, P415 and T1155, were separately purified to homogeneity and characterized.
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Figure 2 depicts the temperature dependence of the apparent specific activity of the mutant enzymes. While T1155 showed a similar temperature dependence to that of the wild-type, P115, P215 and P415 showed the optimum temperatures shifted to lower temperatures. This apparent change of the optimum temperature was caused by the increase in Km at higher temperatures as shown in the following section. The activity of the three mutants was less temperature dependent than that of the wild-type, resulting in higher activity at temperatures below 60°C.
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Table I summarizes the kinetic parameters of the wild-type and cold-adapted mutant IPMDHs at 40 and 70°C. Three of the mutant enzymes, P115, P215 and P415, showed improved kcat values at 40°C. The three mutants can be called kcat-improved cold-adapted mutants. The kcat-improved mutants showed increased kcat values at the cost of the increases in Km for IPM and/or especially NAD (Table I
). This simultaneous increases in kcat and Km values can also be seen in naturally occurring cold-adapted A4 lactate dehydrogenases (Fields and Somero, 1998
) and elastases (Asgeirsson and Bjarnason, 1993
). The kinetic analysis for mutant T1155 revealed the improved binding affinities for both the substrate and NAD (Table I
), resulting in an improvement of the catalytic efficiency (1.3- and 1.8-fold for IPM and NAD, respectively, at 40°C). Low-temperature adapted mutants with improved kcat and Km have been isolated by random mutagenesis (Kano et al., 1997
; Taguchi et al., 1998
, 1999
; Lebbink et al., 2000
; Merz et al., 2000
) or by directed mutagenesis (Imoto et al., 1994
).
It is important to note that NAD is a general cofactor, which is used in many biological reactions in E.coli cells. Accordingly, the concentration of NAD is expected to be constant and independent of the activity of a specific enzyme. At a constant, moderate NAD concentration, higher kcat and lower Km are expected to improve the rate of the catalytic reaction.
Changes in the thermodynamic parameters for the catalytic reaction
The temperature dependence of kcat and Km is shown in Figure 3. The thermodynamic parameters of kcat and Km were derived from the figure and are listed in Table II
. It is clear that kcat is improved by the decrease in
G
that can be ascribed to the lower activation enthalpy
H
in kcat improved mutants (P115, P215, P415). It is interesting that
GKm especially for NAD was significantly increased in the mutants. The increase in
GKm for NAD is induced by the increase in
HKm, although the increase is partially compensated by the increase in
SKm. Relatively temperature-independent catalysis of these kcat-improved mutants can be ascribed to the decreased activation enthalpy. Although there are no reports of thermodynamic parameters for isolated cold-adapted mutant enzymes, a similar observation has been reported for naturally occurring cold-adapted myofibrillar ATP synthases (Johnston and Goldspink, 1975
) and lactate dehydrogenases (Low and Somero, 1974
). The evidence suggests that an increase in catalytic activity at low temperature is induced by a decrease in activation enthalpy in this type of cold adaptation. Consequently, the catalysis becomes less temperature dependent, as seen in the exothermic enzymes.
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Location of the mutation sites in the 3D structure of IPMDH
Residues substituted in cold-adapted mutants are shown on a ribbon model of T.thermophilus IPMDH in Figure 4. T.thermophilus IPMDH binds the extended form of NAD (Hurley and Dean, 1994
). The enzyme interacts with NAD at the adenine moiety: the adenine portion of NAD is in hydrophobic contact with side chains of Ile11, Val15, Leu254, Gly255 and Ile279 and with the aliphatic portion of Asp326 (Hurley and Dean, 1994
). Two mutant enzymes, P115 and P415, possess amino acid substitutions in one of the binding residues, Val15Ile. P415 showed kinetic behavior similar to that of P115, suggesting that the altered kinetic properties in P415 are due at least partially to the substitution Val15Ile. The change of the temperature dependence of the kinetic properties of P115 and P415 mutant enzymes, as well as the low binding affinity toward NAD, can be interpreted as a result of improper fitting of the adenine portion caused by the change of the binding pocket. This interpretation agrees with the results of the kinetic analysis of lactate dehydrogenases of naturally cold-adapted organisms, in which a less temperature-independent catalytic function was ascribed to the decreasing number of weak interactions in the enzymesubstrate complex (Borgmann et al., 1975
).
Another kcat-improved mutant, P215, showed similar kinetic behavior to P115 and P415 (Table I). The substitution is found at Val126 and the site is located in domain II. Crystal structural analysis of the mutant Val126Met revealed that the mutation induced the movement of Leu254 that interacts with the adenine ring (R.Hirose, personal communication).
The mutant T1155 improved the catalytic function by a substitution of Ser92 with phenylalanine. As shown in Figure 4A, the residue is located in helix-d from Pro86 through Lys94 in the active site (Kadono et al., 1995
; Imada et al., 1998
). The substitution of Ser92 seems to induce a geometric change of the binding residues for the substrates. However, the detailed mechanism for the increased affinity of the Km-improved mutants with the coenzyme is not clear.
Thermal stability of the cold-adapted mutant IPMDHs
The thermal stability of the enzymes was investigated by two methods. Irreversible thermal inactivation was analyzed by measuring residual activity after heat treatment (Figure 5A). Half inactivation temperatures (T1/2) were 87°C (wild-type), 85°C (P115), 87°C (P215), 82°C (P415) and 87°C (T1155). Temperature-dependent unfolding of the secondary structure was followed by monitoring changes in circular dichroism (CD) at 222 nm (Figure 5B
). Half denaturation temperatures (T1/2) monitored by CD were 87, 85, 87, 80 and 87°C for wild-type, P115, P215, P415 and T1155 enzymes, respectively and were similar to those estimated from the residual activity measurement. These two analyses revealed that two of the cold-adapted mutant IPMDHs, P215 (kcat-improved type) and T1155 (Km-improved type), retained the original thermal stability, whereas P115 and P415 IPMDHs slightly lost stability. A kcat-improved mutant with the same thermal stability as the wild-type has been reported for P.furiosus ß-glucosidase (Lebbink et al., 2000
). A Km-improved mutant with the same thermal stability as the original enzyme has been reported for subtilisin (Kano et al. 1997
). These results, including ours, suggest that activity can be improved without loss of thermal stability, supporting the idea that thermal stability and activity at moderate temperature are partially independent factors (Imoto et al., 1994
; Akanuma et al., 1998
; Giver et al., 1998
).
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Cold-adapted mutants were efficiently isolated from a thermophilic enzyme by laboratory evolution. The laboratory evolution allowed us to identify mutation sites to improve catalytic activity without lowering thermal stability. The strategy described here provides a general way of creating active enzymes at lower temperatures with high thermal stability.
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Notes |
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5 To whom correspondence should be addressed. E-mail: yamagish{at}ls.toyaku.ac.jp
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Acknowledgments |
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Received October 6, 2000; revised November 27, 2000; accepted December 1, 2000.