Protein Engineering, Vol. 15, No. 1, 35-41,
January 2002
© 2002 Oxford University Press
An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution
1 Department of Applied Biological Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, 2 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 and 3 Department of Bioscience and Biotechnology, Aomori University, Aomori 030-0943, Japan
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Abstract |
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Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in the absence of Fru(1,6)P2 under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P2-binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P2 or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P2 under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P2-independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P2 requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P2, through a slight decrease and increase of the Fru(1,6)P2 sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P2. The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.
Keywords: allosteric enzyme/fructose 1,6-bisphosphate/L-lactate dehydrogenase/lactic acid bacteria/Lactobacillus casei
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Introduction |
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Bacterial L-lactate dehydrogenases (L-LDHs) (EC 1.1.1.27) exhibit a wide variety of catalytic properties (Garvie, 1980
), together with high amino acid sequence divergence (Griffin et al., 1992). Whereas vertebrate cells contain non-allosteric L-LDH isozymes (Holbrook et al., 1975), many bacterial cells possess allosteric L-LDHs, which usually require fructose 1,6-bisphosphate [Fru(1,6)P2] for their activities (Garvie, 1980). Through analysis based on protein engineering, the allosteric regulation of L-LDHs has been extensively studied for Thermus caldophilus (Matsuzawa et al., 1988; Schröder et al., 1988; Koide et al., 1991, 1992), Bacillus stearothermophilus (Clarke et al., 1987, 1989; Wigley et al., 1992; Cameron et al., 1994) and Bifidobacterium longum (Iwata et al., 1994; Fushinobu et al., 1996, 1998) allosteric enzymes. Whereas these enzymes exhibit their catalytic activities even in the absence of Fru(1,6)P2, some L-LDHs appear to absolutely require Fru(1,6)P2 for their enzyme activities (Götz and Schleifer, 1975; Garvie, 1980), although little is known about the structure–function relationship of these enzymes.
Among known L-LDHs, Lactobacillus casei L-LDH shows unique allosteric properties (Holland and Pritchard, 1975; Hensel et al., 1977, 1983; Garvie, 1980). Whereas the enzyme absolutely requires Fru(1,6)P2 for its catalytic activity under neutral conditions, it exhibits marked catalytic activity even in the absence of Fru(1,6)P2 under acidic conditions, where the enzyme gives sigmoidal and hyperbolic saturation curves for substrate pyruvate without and with Fru(1,6)P2, respectively. Kinetic studies (Gordon and Doelle, 1976; Hensel et al., 1983) and affinity labeling with 3-bromopyruvate (Hensel et al., 1983) have revealed that pyruvate is bound to an allosteric binding site, which corresponds to the Fru(1,6)P2-binding site, when Fru(1,6)P2 is absent.
Unlike the L.casei enzyme, the L-LDH of Lactobacillus pentosus, previously called Lactobacillus plantarum, is a non-allosteric enzyme, like the vertebrate enzymes (Taguchi and Ohta, 1992; Arai et al., 2001; Uchikoba et al., 2002), although the two Lactobacillus enzymes show particularly high amino acid sequence identity (Taguchi and Ohta, 1991, 1992; Griffin et al., 1992). Among the 10 residues of the Fru(1,6)P2-binding site, the L.casei and L.pentosus enzymes only differ in the amino acid residues at positions 188 and 205 (residues are numbered according to Eventoff et al., 1977), the former enzyme possessing His residues, but the latter Asp and Thr, respectively (Taguchi and Ohta, 1992). The site-specific replacement of His188 with Asp completely abolishes the heterotropic effect of Fru(1,6)P2 on the L.casei enzyme (Taguchi and Ohta, 1995), indicating that His188 is essential for the regulation by Fru(1,6)P2, as in the cases of the well studied Fru(1,6)P2-regulated L-LDHs (Schröder et al., 1988; Clarke et al., 1989; Iwata et al., 1994). In this work, we describe the replacement of His205 with Thr, which did not induce a marked reduction in the enzyme activation by Fru(1,6)P2, but a great loss of the Fru(1,6)P2-independent activation of the enzyme by a substrate, in contrast with the case of His188 replacement.
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Materials and methods |
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Preparation of the wild-type and mutant L.casei L-LDHs
Oligodeoxynucleotide 5'-GAT GTT AGC AGT AGC TTG CCA TAC AGG-3' was purchased from Takara Syuzo and used to replace His205 with Thr. Site-directed mutagenesis was performed with a MUTA-GENE in vitro mutagenesis kit (Bio-RAD) according to Kunkel (Kunkel, 1985). Cultivation of recombinant Escherichia coli cells harboring expression plasmids for the gene encoding L.casei IAM 12473 (=ATCC 393) wild-type L-LDH, and H188D and H205T mutant L-LDHs, in which His188 and His205 are replaced with Asp and Thr, respectively, and purification of these three types of enzyme were performed essentially as described previously (Taguchi and Ohta, 1995). The purity of the enzyme preparations was examined by SDS–PAGE according to Laemmli (Laemmli, 1970). The enzyme samples for chemical modification with 3-bromopyruvate were confirmed to be coenzyme-free by measurement of the absorbance at 260 and 280 nm before use.
Enzyme assay and protein determination
The assaying of the purified enzymes was carried out at 30°C in 50 mM sodium acetate buffer (pH 5.0) or 50 mM sodium morpholinepropanesulfonic acid (MOPS) buffer (pH 7.0) containing 0.1 mM NADH. One unit was defined as the catalytic rate of the conversion of 1 µmole of substrate per minute. Protein concentrations were determined with Bio-RAD Protein Assay protein reagent (Bio-RAD), using bovine serum albumin as a standard protein. Since the catalytic reaction with the L.casei enzyme in the presence of Fru(1,6)P2 was markedly inhibited by high concentrations of substrates, kinetic parameters such as KM and maximal velocity (Vmax) were estimated using only data obtained with substrate concentrations sufficiently low and non-inhibitory to give linear lines on double-reciprocal plotting. For the reactions that showed significant cooperativity, the Hill equation (Dixon and Webb, 1979) was used for sigmoidal curve fitting to obtain kinetic parameters such as the Hill coefficient (h) and half-saturating substrate concentration (S0.5). Inhibition constants for phosphoenolpyruvate, inorganic phosphate and 2-ketoglutarate were determined essentially according to a previous report (Hensel et al., 1983).
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Results |
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Effects of His205 replacement on the homotropic and heterotropic enzyme activation at pH 5.0
Unlike the wild-type L.casei L-LDH, the H205T mutant enzyme, in which His205 is replaced with Thr, exhibited no marked catalytic activity in the absence of Fru(1,6)P2 at pH 5.0, even when the pyruvate concentration was increased to 50 mM (Figure 1). Nevertheless, the reaction with the H205T enzyme was greatly enhanced in the presence of 5 mM Fru(1,6)P2, the shape of the saturation curve being hyperbolic for pyruvate, as in the case of the wild-type enzyme. The kinetic parameters of the enzymes for pyruvate saturation are summarized in Table I, together with those of the wild-type enzyme and the H188D enzyme, in which His188 is replaced with Asp. In the presence of 5 mM Fru(1,6)P2, the H205T enzyme exhibited a slightly increased KM value and virtually the same Vmax value compared to the wild-type enzyme, whereas the activation effect of Fru(1,6)P2 was lost in the H188D enzyme.
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As in the case of the wild-type enzyme, the reaction of the H205T enzyme in the presence of 5 mM Fru(1,6)P2 was markedly inhibited at high concentration of substrate. Such a substrate inhibition is generally observed in L-LDHs, and is thought to be a consequence of the formation of an aduct between pyruvate and NAD+, before NAD+ is released from the enzyme (after lactate is released) (Eszes et al., 1996
; Hewitt et al., 1999). When the data were simply interpreted with the equation for the substrate inhibition: 
, where [S] is the substrate concentration and v is the velocity at substrate concentration [S], according to Eszes et al. (Eszes et al., 1996), the inhibition constants for the wild-type and H205T enzymes were determined to be 62 and 90 mM, respectively.
It is known that pyruvate analogs instead of Fru(1,6)P2 also stimulate the reaction with L.casei L-LDH under acidic conditions (Hensel et al., 1983). In the case of the wild-type or H188D enzyme, two pyruvate analogs, oxamate and 2-ketoglutarate, apparently stimulated the catalytic activity by reducing the homotropic cooperativity, and the latter also induced a marked increase in the Vmax value, beside significant reduction of the S0.5 and h values (Figure 1
and Table I). In contrast to these two types of enzymes, the H205T enzyme exhibited too low activity in the presence of 10 mM oxamate or 2-ketoglutarate to determine the exact kinetic parameters. Figure 2 shows the activities of the H205T enzyme at pH 5.0 in the presence of various concentrations of oxamate and 2-ketoglutarate with a fixed concentration of pyruvate (1 mM), together with the activity of the wild-type enzyme. Oxamate exhibited only a slight activation effect on the H205T mutant enzyme compared to the wild-type enzyme, and 2-ketoglutarate had no marked effect on the H205T enzyme up to 50 mM, in contrast to the case of the wild-type enzyme.
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Effects of His205 replacement on the catalytic properties at pH 7.0
It is known that L.casei L-LDH absolutely requires Fru(1,6)P2 for its enzyme activity under neutral conditions, where the activation function of Fru(1,6)P2 is markedly enhanced in the presence of certain divalent metal ions such as Mn2+ (Holland and Pritchard, 1975; Gordon and Doelle, 1976; Hensel et al., 1977). As in the case of the wild-type enzyme, the H205T enzyme exhibited no detectable catalytic activity at pH 7.0 in the absence of Fru(1,6)P2, but significant activity in the presence of 5 mM Fru(1,6)P2, the pyruvate saturation curve being sigmoidal (Figure 3). In addition, MnSO4 (10 mM) also stimulated the catalytic reaction with the H205T enzyme in the presence of 5 mM Fru(1,6)P2, the sigmoidal pyruvate saturation curve changing into a hyperbolic one. The kinetic parameters of these enzymes at pH 7.0 are also shown in Table I. The H205T enzyme exhibited essentially the same catalytic properties as the wild-type enzyme, and similar kinetic parameters, i.e. S0.5, h and Vmax values, to those of the wild-type enzyme, unlike in the case of pH 5.0.
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Effects of His205 replacement on the pH-dependent Fru(1,6)P2 sensitivity
The activation function of Fru(1,6)P2 as to the H205T enzyme was evaluated at pH 5.0 and 7.0 in the presence of 1.0 and 10 mM pyruvate, respectively (Figure 4 and Table II). At pH 5.0, the wild-type L.casei enzyme was much more (~2.5x 104-fold) sensitive to Fru(1,6)P2 than at pH 7.0, the S0.5 for Fru(1,6)P2 being 2 µM (Table II). The H205T enzyme also exhibited high sensitivity to Fru(1,6)P2 at pH 5.0, but required a slightly higher (4-fold) concentration of Fru(1,6)P2 for activation than the wild-type enzyme. Like the wild-type enzyme, the H205T enzyme also required a much higher concentration of Fru(1,6)P2 for the activity at pH 7.0, when MnSO4 was absent. In this case, however, the H205T enzyme exhibited slightly higher sensitivity (a 2.5-fold lower S0.5 value) to Fru(1,6)P2 than the wild-type enzyme, unlike in case of pH 5.0. Consequently, the H205T enzyme exhibited only a 2.5x103-fold lower (10-fold different from the case of the wild-type enzyme) Fru(1,6)P2 sensitivity at pH 7.0 than at pH 5.0. As in the case of the wild-type enzyme, the activation function of Fru(1,6)P2 at pH 7.0 with the H205T enzyme was markedly improved by 10 mM MnSO4.
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Effects of His205 replacement on 3-bromopyruvate-induced desensitization to Fru(1,6)P2
It is known that 3-bromopyruvate specifically modifies His188 of L.casei L-LDH in the absence of the coenzyme NADH, and thereby leads to desensitization of the enzyme to Fru(1,6)P2, indicating that pyruvate or an analog binds to the allosteric site (Hensel et al., 1983). Therefore, to evaluate the effect of the replacement of His205 on the pyruvate binding to the allosteric site, the wild-type and H205 mutant enzymes were treated with 10 mM 3-bromopyruvate with and without 10 mM oxamate (Figure 5). As in the case of the wild-type enzyme, 3-bromopyruvate also induced marked desensitization of the H205T enzyme to Fru(1,6)P2. The decrease in the Fru(1,6)P2-sensitive enzyme indicated an apparent first-order reaction, and gave the same half-life of 2.1 h for the wild-type and H205T enzymes in the absence of oxamate. In the presence of 10 mM oxamate, the desensitization was significantly inhibited in the cases of both the wild-type and H205T enzymes, although the latter enzyme had a slightly shorter half-life (2.7 h) than that of the former enzyme (3.3 h).
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Effects of His205 replacement on the effects of inhibitors that compete with Fru(1,6)P2
It has been reported that phosphoenolpyruvate and 2-ketoglutarate competitively inhibit the activation function of Fru(1,6)P2 as to the wild-type L.casei L-LDH under acidic conditions, whereas they exhibit weak but significant activation effects in the absence of Fru(1,6)P2 (Hensel et al., 1983). As in the case of the wild-type enzyme, both phosphoenolpyruvate and 2-ketoglutarate exhibited marked inhibitory effects on the H205T enzyme through competition with Fru(1,6)P2, and gave only slightly increased (2.4- to 2.3-fold, respectively) inhibition constants (KI) as compared with the case of the wild-type enzyme (Table III); however, they exhibited virtually no activation effects on the H205T enzyme in the absence of Fru(1,6)P2, unlike in the case of the wild-type enzyme. On the other hand, as compared with these two inhibitors, inorganic phosphate is a more general inhibitor competing with the activation by Fru(1,6)P2 of many Fru(1,6)P2-dependent L-LDHs (Garvie, 1980) including the L.casei enzyme (Gordon and Doelle, 1976). Inorganic phosphate also exhibited an inhibitory effect on the H205T enzyme as in the case of the wild-type enzyme, but gave a slightly less increased KI as compared with phosphoenolpyruvate and 2-ketoglutarate (Table III).
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Comparison of amino acid residues of the Fru(1,6)P2-binding sites
Table IV shows a comparison of the 10 amino acids comprising the Fru(1,6)P2-binding site, or the corresponding site in the case of non-allosteric enzymes, among L-LDHs of L.casei-related lactic acid bacteria and other representative L-LDHs. As compared with Arg173 and His188, which are essential for the regulation by Fru(1,6)P2 of general Fru(1,6)P2-activated L-LDHs (Clarke et al., 1987, 1989; Matsuzawa et al., 1988; Schröder et al., 1988), His205 is poorly conserved in known allosteric and non-allosteric L-LDHs, and is actually missing in the well studied enzymes such as the B.stearothermophilus, T.caldophilus and B.longum enzymes, although the latter two are activated through the cooperative effects of substrates in the absence of Fru(1,6)P2 (Taguchi et al., 1984; Matsuzawa et al., 1988; Fushinobu et al., 1996, 1998), as in the case of the L.casei L-LDH. On the other hand, the amino acids of the allosteric site are well conserved in the enzymes of some lactic acid bacteria related to L.casei, as compared with the case of other L-LDHs (Table IV). As well as His188, His205 is conserved in the Fru(1,6)P2-dependent enzymes of Lactococcus lactis, Streptococcus mutans and Strepococcus thermophilus enzyme, which are Fru(1,6)P2-dependent L-LDHs except for the last one (Ito and Sasaki, 1994). In contrast, the L.pentosus (Taguchi and Ohta, 1992), L.plantarum (Ferain et al., 1994) and Pediococcus acidilactici (Garmyn et al., 1995) L-LDHs, which constitute a group of non-allosteric L-LDHs, specifically lack both His205 and His188.
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Discussion |
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As in the case of vertebrate L-LDHs (Holbrook et al., 1975
), known bacterial Fru(1,6)P2-dependent L-LDHs are tetrameric enzymes, and the four subunits of the enzymes are related through three molecular two-fold axes named P, Q and R (Wigley et al., 1992; Iwata and Ohta, 1993; Iwata et al., 1994). It is also known that the Fru(1,6)P2-binding (allosteric) site, which corresponds to the anion-binding site in vertebrate enzymes, is located on the P-axis subunit interface of allosteric L-LDHs, and one site is composed of residues from two subunits of one P-axis dimer in the case of the known allosteric L-LDHs (Figure 6). Although His205 is located in the allosteric site of the L.casei enzyme, it is unlikely that it directly participates in Fru(1,6)P2 binding, unlike in the case of His188, because its replacement with Thr induces only a 4-fold decrease and even a 2.5-fold increase in Fru(1,6)P2 sensitivity at pH 5.0 and 7.0, respectively (Figure 4 and Table II). In the case of the B.stearothermophilus (Wigley et al., 1992) and B.longum (Iwata et al., 1994) L-LDHs, the amino acid at position 205 (Gln and Ser, respectively) is actually too far from the bound Fru(1,6)P2 molecule to undergo a direct interaction with it, whereas His188 directly interacts with the Fru(1,6)P2 molecule (Figure 6).
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Nevertheless, it is notable that the replacement of His205 significantly affected the pH dependence of the Fru(1,6)P2 sensitivity, through a reduction and increase in the Fru(1,6)P2 sensitivity at pH 5.0 and 7.0, respectively (Figure 4
and Table II). Allosteric L-LDHs usually exhibit a marked pH dependence in their Fru(1,6)P2 sensitivity, and it is suggested that His188 is pivotal in such pH dependence, since it is more deprotonated and unfavorable for the interaction with Fru(1,6)P2 under higher pH conditions (Clarke et al., 1987). Furthermore, in the case of the L.casei enzyme, which shows a particularly high pH-dependent sensitivity to Fru(1,6)P2, (Mayr et al., 1982) our results indicate that His205 is also, although indirectly, largely involved in the pH dependence of the Fru(1,6)P2 sensitivity (Figure 4 and Table II). Thus, His205 replacement did not markedly impair the heterotropic effect of Fru(1,6)P2 or Mn2+, but greatly diminished the activating effects of substrates (analogs) in the absence of Fru(1,6)P2, leading to an absolute Fru(1,6)P2 requirement for the enzyme activity of the L.casei L-LDH, in contrast to the case of the replacement of His188. This suggests that only a small structural change such as a single amino acid replacement may sufficiently change such absolute or non-absolute Fru(1,6)P2 dependence of allosteric L-LDHs.
In the case of the B.longum alosteric L-LDH, it has been reported that the allosteric transition can be assumed as two states, the inactive (T) and active (R) states, according to the concerted model demonstrated by Monod et al. (Monod et al., 1965), and the allosteric and catalytic sites communicate with each other through the quaternary structure change (Iwata et al., 1994; Fushinobu et al., 1996, 1998). In the concerted model, a homotropic regulation for allosteric L-LDHs is described by the equation:
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), this equation well fitted the data for the pyruvate saturation at pH 5.0 without Fru(1,6)P2 (Figure 1) with a KT value of 88.8 (±18.3) mM and L value of 6.48 (±0.72)x105 for the wild-type enzyme, and a KT value of 35.7 (±3.1) mM and L value of 4.98 (±0.27)x104 for the H188D enzyme. Although the H205T enzyme exhibits much weaker activity in the absence of Fru(1,6)P2, its KR and Vmax values may approximate to the values for the wild-type enzyme, since these two enzymes exhibit virtually the same activity when they are fully activated by a sufficient concentration of Fru(1,6)P2 (Table I). From this approximation, the L value of H205T enzyme was estimated to be higher than 108, even when it was assumed that pyruvate was bound to the R state exclusively. This suggests that His205 at least 100-fold stabilizes the R state of the L.casei enzyme at pH 5.0, whereas such a stabilization effect is not evident at pH 7.0, where the wild-type and H205T enzymes exhibit virtually the same properties (Figure 3). In addition, the KT value for the H205T was estimated to be much higher (more than 1000 mM) than the value for the wild-type enzyme, even if L = 
was assumed. The above simple equation for homotropic activation lacks factors such as the pyruvate binding to the allosteric site, which may partially stabilize the R state of L.casei L-LDH (Hensel et al., 1983). The difference in the apparent KT values suggest that His205 may be also involved in the allosteric effect of this substrate binding, although its replacement only slightly changed the apparent substrate (analogs) binding to the allosteric site (Table III and Figure 5).
The three-dimensional structures of the T and R states of the B.longum enzyme (Iwata et al., 1994) imply that one of the potent triggers for the Fru(1,6)P2-induced enzyme activation is the strong ionic interaction between the Arg173 guanidino group and the Fru(1,6)P2 phosphate moiety, in which the motion of Arg173 to the Fru(1,6)P2 phosphate coordinates the allosteric change of the enzyme conformation with the R state. Figure 6 shows the allosteric sites of the T and R states of the B.longum L-LDH (A), together with that of the Fru(1,6)P2-bound B.stearothermophilus enzyme (Wigley et al., 1992) and the corresponding site for the non-allosteric L.pentosus (Uchikoba et al., 2001) enzyme (B), both of which can be considered to be in the R state. As in the case of Arg173, the amino acid at position 205 of the B.longum enzyme (Ser205) also significantly moves across the P-axis dimer interface during the allosteric transition of the B.longum enzyme, suggesting that this residue may also potentially trigger the allosteric transition through the formation of some interaction networks. Since the Fru(1,6)P2-independent activation by a substrate highly depends on acidic conditions, only the protonated form of His205 is possibly able to participate in such interaction networks.
Although one of the possible ligands for His205 is a pyruvate molecule bound to the allosteric site, it is not evident as to whether or not His205 directly interacts with pyruvate in the allosteric site. The H205T enzyme was desensitized to Fru(1,6)P2 by 3-bromopyruvate, and protected from the desensitization by oxamate in a similar way to the case of the wild-type enzyme (Figure 5). In addition, the mutant enzyme exhibited only slightly increased KI values for pyruvate analogs that compete with Fru(1,6)P2 (Table III). These results indicate that His205 is not essential for the substrate binding to the allosteric site, at least when the enzyme is in the T state conformation, which should have been predominant in these experiments.
Another possible ligand for His205 is some amino acid residue(s) of the protein, possibly on the P-axis interface of the counterpart subunit related through the P-axis, toward which His205 appears to move during the conformational change to the R state (Figure 6). Using X-ray crystallography of several types of L.casei L-LDH, we have found that the imidazole of His205 can form an ionic hydrogen bond with amino acids, such as Glu211, on the counterpart subunit of the P-axis dimer (K.Arai et al., unpublished results). In any case, it is likely that the protonated His205 can participate in some interaction network that stabilizes the R state of the enzyme, and thereby stimulates the enzyme activation under acidic conditions. Since substrate pyruvate has a much weaker activation function than Fru(1,6)P2, such a His205-related interaction network may be particularly important in the case of Fru(1,6)P2-independent activation. It is interesting that His205 is well conserved in the enzymes of lactic acid bacteria related to L.casei (Table IV). We are currently investigating the crucial role of His205 through detailed X-ray crystallography of and additional amino acid replacements in the L.casei enzyme.
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Notes |
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4 To whom correspondence be addressed. E-mail: htaguchi{at}rs.noda.sut.ac.jp
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Scientific Research to H.T. from the Ministry of Education, Science, Sports and Culture of Japan.
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Received July 19, 2001; revised October 3, 2001; accepted October 16, 2001.
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