Central Research Laboratories and 1 Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho Kawasaki-ku, Kawasaki 210-8681 and 3 Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurosawa, Kosugi, Toyama 939-0398, Japan
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Abstract |
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Keywords: acid phosphatase/crystal structure/nucleotide/protein engineering/site-directed mutagenesis
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Introduction |
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Some bacterial class A non-specific acid phosphatases (NSAPs) exhibit phosphotransferase activity in addition to their intrinsic phosphatase activity (Ishikawa et al., 2000; Mihara et al., 2000
, 2001
). The phosphotransferase activity of Morganella morganii NSAP (MM-NSAP) was exploited to produce a 5'-nucleotide by the selective phosphorylation of a nucleoside in the C-5' position using pyrophosphate (PPi), as shown in Scheme 1 (Asano et al., 1999a
,b
). The regioselective phosphotransferase activity is considered as one of the merits of the enzyme when it is used in the nucleoside phosphorylation process.
Unfortunately, the phosphatase activity of the wild-type NSAPs dominates the phosphotransferase activity, and consequently, a considerable amount of the 5'-IMP synthesized by the phosphotransferase activity is dephosphorylated into inosine by the dominating phosphatase activity. However, if the phosphotransferase activity is enhanced in comparison to the phosphatase activity, then the process using the NSAPs could be useful in industry and replace the current chemical (Yoshikawa et al., 1969) and enzymatic (Mori et al., 1997
) processes to produce 5'-IMP. Mihara et al. (Mihara et al., 2000
) randomly mutated MM-NSAP and successfully generated the G74D/I153T mutant with enhanced PPi-inosine phosphotransferase activity, which is accounted for by the reduction in the Km value for inosine: from 117 (wild-type) to 42 mM (G74D/I153T). However, the structureactivity relationship of the improved enzyme has remained unexplained.
| (Scheme 1) |
Bacterial class A NSAPs with regioselective phosphotransferase activity have also been isolated from Escherichia blattae (Ishikawa et al., 2000), Enterobacter aerogenes, Providensia stuartii, Klebsiella planticola (Mihara et al., 2001
), and so on. Mihara et al. (Mihara et al., 2001
) investigated the phosphotransferase activity of these NSAPs and found that the amounts of 5'-IMP produced by the NSAPs are closely related to their Km values for inosine. This finding is plausible, since the PPi-inosine phosphotransferase activity prevails over the phosphatase activity if the phospho-enzyme intermediate prefers an attack by inosine to that by water.
We have embarked on research to understand the structureactivity relationship of the improved enzyme and to obtain an even more efficient enzyme by rational site-directed mutagenesis, since further improvement of the enzyme by random mutation seemed to be difficult. We selected the E.blattae NSAP (EB-NSAP) as the target of rational mutagenesis, despite its rather higher Km value (200 mM) for inosine, since the crystal structure of the enzyme has been determined at 1.9 Å resolution (Ishikawa et al., 2000), while no structural data are available for the other NSAPs. EB-NSAP is a 150 kDa homohexamer, and has 77% sequence identity to MM-NSAP. Since the crystal structure of EB-NSAP complexed with the transition-state analog, molybdate, has also been solved (Ishikawa et al., 2000
), a hypothetical binding mode of inosine to the phospho-enzyme intermediate was modeled using the enzymemolybdate complex structure. First, the G74D and I153T mutations were introduced to EB-NSAP, since the previous random mutagenesis of MM-NSAP showed that these mutations were effective in reducing the Km value for inosine (Mihara et al., 2000
). Subsequent rational site-directed mutagenesis based on the substrate-binding model yielded an industrially viable mutant, S72F/G74D/I153T, which has a 5.4-fold improvement in the Km value for inosine, as compared to the wild-type. Our results demonstrate that the combination of random mutagenesis and rational site-directed mutagenesis can be a powerful tool to drastically alter a property of an enzyme. In this article, we compare and interpret the G74D/I153T and wild-type structures, and describe how the rational site-directed mutagenesis was performed.
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Materials and methods |
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Crystallization of G74D/I153T was performed using the vapor diffusion method in hanging drops at 20°C. Crystals were grown from drops containing protein (20 mg/ml, 20 mM Tris pH 8.0) and reservoir (38% PEG400, 20 mM TrisHCl pH 8.4) in a 1:1 ratio. Crystals appeared after a few days and grew to a maximum size of 0.7x0.4x0.2 mm within a week. The crystals belong to the space group P212121 with unit cell dimensions a = 137.8 Å, b = 168.2 Å, c = 58.4 Å, and with a hexamer in the asymmetric unit, giving a calculated value of 64% for solvent content. The best data set was collected at 15°C using the macromolecular-oriented Weissenberg camera (Sakabe, 1991) installed at beamline 6B of the Synchrotron Radiation Source at the National Laboratory of High Energy Physics (Photon Factory). Measured intensities were integrated using WEIS (Higashi, 1989
), and were scaled with SCALA and AGROVATA (Collaborative Computing Project No. 4, 1994
). The structure of G74D/I153T was solved by molecular replacement using AMORE (Collaborative Computing Project No. 4, 1994
). The coordinates of the hexameric structure of the wild-type enzyme (Ishikawa et al., 2000
) were used as a search model. Using data in the range of 103 Å, the correct rotation and translation solution gave an initial R factor of 0.38. The structure was refined by iterative cycles of manual rebuilding using QUANTA (MSI) and refinement using CNX (Brünger et al., 1987
), using data to 2.5 Å. The non-crystallographic restraints were applied in the refinement to suppress an over-fitting of data. The final R factor and Rfree are 0.242 and 0.285, respectively, for 45 889 unique reflections in the 102.5 Å shell (97.5% complete). The r.m.s.d.s for bond distances and angles were 0.007 Å and 1.31°, respectively. The final model contains Gly7Asn133 and Lys139His228 of molecule A, Thr6Thr134 and Ser141Lys230 of molecule B, Thr6Thr134 and Ser141Gln227 of molecule C, Gly7Asn133 and Ser141His228 of molecule D, Gly7Asn133 and Ser141Gln227 of molecule E, Thr6Thr134 and Ser141His228 of molecule F, and 371 water molecules.
DNA mutagenesis and production of mutated NSAPs
All basic recombinant DNA procedures, such as isolation and purification of DNA, restriction enzyme digestion, and transformation of Escherichia coli, were performed as described by Sambrook et al. (Sambrook et al., 1989). Mutagenesis was carried out on the E.blattae acid phosphatase gene, cloned in pUC18 (pEAP320) (Ishikawa et al., 2000
). Site-directed mutagenesis was accomplished using the Quick Change Mutagenesis kit (Stratagene) according to its protocol. Mutations were confirmed by sequencing by the dideoxynucleotide chain termination method with a Dye Terminator Cycle sequencing kit (Perkin-Elmer) and a DNA sequencer (model 373A, Perkin-Elmer). Synthesized universal and fragment-specific oligonucleotides for each clone were used as primers.
Escherichia coli JM109 (Yanisch-Perron et al., 1985) was used as the host strain, and LuriaBertani medium (Sambrook et al., 1989
) containing ampicillin (50 µg/ml) was used for the culture of E.coli transformants. Escherichia coli JM109 transformants harboring each plasmid were cultured and harvested as described previously (Mihara et al., 2000
). A crude extract was prepared from each harvested culture as described previously (Asano et al., 1999a
). The G74D/I153T and S72F/G74D/I153T mutant EB-NSAPs were further purified by ammonium sulfate fractionation and ion-exchange, hydrophobic, and gel-filtration column chromatographies as described previously (Asano et al., 1999a
).
Enzyme assay
Phosphotransferase activity was assayed in a standard reaction mixture containing 100 µmol of sodium acetate buffer (pH 5.0), 40 µmol of inosine, 100 µmol of tetrasodium pyrophosphate, and the enzyme solution in a total volume of 1 ml. The reaction mixture was incubated for 10 min at 30°C and then the reaction was stopped by adding 0.2 ml of 2 N HCl. Quantitative determination of inosine and 5'-IMP was carried out by HPLC as described previously (Asano et al., 1999). One unit of phosphotransferase activity was defined as the amount of enzyme that produces 1 µmol of 5'-IMP per minute under the assay conditions. For the determination of Km and Vmax values, assays were carried out at various concentrations of inosine whilst keeping pyrophosphate at large excess levels (100 mM). As the solubility of inosine was limited, kinetic constants for inosine were determined with the substrate concentration range from 0.5 to 100 mM. Each assay was done once and the data were analyzed using a HanesWoolf plot.
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Results and discussion |
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The bound molybdate in the enzymemolybdate complex structure was replaced by phosphate, and the inosine was placed on a depression surrounded by Leu16, Ser71, Ser72 and Glu104, with the 5'-O atom of the ribose situated close to the phosphorus. As EB-NSAP is not specific to inosine, it was not possible to place the inosine with a unique conformation. However, the position of the phosphate and the structure around it indicated that the ribose must be situated near His150. Although the position of the base is affected by the conformation and the position of the ribose, it seems most plausible to place it near Ser71 and Glu104. One of the possible inosine-binding modes is shown in Figure 1. In this model, Ile153 is situated close to the ribose, and therefore this residue can be considered as a part of the inosine-binding site. An automatic docking of the inosine to EB-NSAP has been hampered by lack of force field parameters for the covalent phosphohistidine. The development of empirical force constants for the phosphoimidazole is in progress.
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Before starting rational mutagenesis based on the enzymeinosine-binding model, the G74D and I153T mutations were introduced to EB-NSAP, since the previous random mutagenesis of MM-NSAP showed that these two mutations enhanced the phosphotransferase activity (Mihara et al., 2000). The productivity of wild-type EB-NSAP was very low, and only 13.6 g/l 5'-IMP was produced with a maximum molar yield of only 22% from inosine using E.coli JM109 overproducing the wild-type enzyme (Mihara et al., 2001
). In contrast, the productivity of the resultant EB-NSAP mutant was greatly improved, as expected. Using E.coli JM109 overproducing G74D/I153T, 103 g/l 5'-IMP was produced with a molar yield of 52% from inosine, and the dephosphorylation of the produced 5'-IMP was considerably depressed (Figure 2
).
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The wild-type and G74D/I153T proteins crystallized in different crystal forms, although only two residues are different. The asymmetric unit of the wild-type crystal has one subunit, while that of the mutant crystal accommodates one hexamer. Despite the different crystal forms, the overall structure of G74D/I153T is essentially identical to that of the wild-type, as revealed by the r.m.s.d.s between the equivalent C positions of 0.300.32 Å, calculated when the wild-type subunit structure was superimposed with each of the six subunits of G74D/I153T, using 214 C
atoms (Gly7Asn133 and Ser141Gln227).
In the EB-NSAP structure, Gly74 and Ile153 are situated on the 4-helix and the
10-helix, respectively (Ishikawa et al., 2000
). In the G74D/I153T structure, the carboxyl group of Asp74 forms a hydrogen bond with the O
of Ser71, and the side-chain O
of Thr153 hydrogen bonds to the main-chain carbonyl oxygen of Ile103 (Figure 3
). Despite these newly formed hydrogen bonds, the overall structure of G74D/I153T remains unchanged. This structural similarity clearly indicates that the difference in the Km value can be ascribed to the local structural differences around the two mutated residues.
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Site-directed mutagenesis for a reduced Km value
While building the inosine-binding model, we noticed that there is little interaction between the enzyme and the inosine base. In order to introduce baseenzyme interactions, we searched for residues whose replacements with aromatic residues would possibly lead to the formation of aromaticaromatic interactions without perturbing the enzyme structure. Such interactions between the base of a nucleotide/nucleoside and the side chain of an aromatic residue are observed in most nucleotide/nucleoside-recognizing enzymes, such as ribonuclease T1 (Arni et al., 1988) and purine nucleoside phosphorylase (Koellner et al., 1998
). Consequently, Leu16, Ser71, Ser72 and Glu104 were chosen as candidates for replacement. A typical aromaticaromatic interaction between residues, such as phenylalanine, tyrosine and tryptophan, has a non-bonded potential energy of between -1 and -2 kcal/mol, which was determined from 34 protein structures from the Protein Data Bank (Burley and Petsko, 1985
). To maximize the possibility of the formation of an aromaticaromatic interaction, the introduction of the largest amino acid, tryptophan, to the above four positions seemed promising, and thus the following four mutations were designed: Leu16
Trp, Ser71
Trp, Ser72
Trp and Glu104
Trp. The Km values and the relative activities of crude extracts of the resultant mutants are shown in Table II
. Among the four mutants, S72W/G74D/I153T had the lowest Km value. In addition, this mutant showed higher activity than that of the parent. In the inosine-binding model, the introduced tryptophan side chain at position 72 is not close enough to the inosine base to make an ideal aromaticaromatic interaction; the distance between them is estimated to be 5.5 Å. However, it is likely that the loop containing Trp72 undergoes a slight conformational change upon inosine binding, and thus an ideal aromaticaromatic interaction is formed, because this loop is relatively flexible and significantly changes its conformation upon the formation of the phospho-enzyme intermediate (Ishikawa et al., 2000
). Actually, when the structures of the enzymemolybdate complex and the native enzyme are superimposed, Ser72 in the native enzyme structure is situated very close to Ser71 in the enzymemolybdate complex structure, which is located near the inosine base in the enzymeinosine-binding model (Figure 1
).
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To characterize S72F/G74D/I153T further, we purified the mutant protein and measured its kinetic parameters (Table I and Figure 4
). The Km and Vmax values of the mutant were 2.9-fold lower and 2.7-fold higher than those of the parent, respectively. Next, the time course of 5'-IMP production using E.coli JM109 overproducing the mutant was measured (Figure 2
). The productivity of the S72F/G74D/I153T mutant was superior to that of the G74D/I153T mutant, and 140 g/l of 5'-IMP were obtained with a molar yield of 71% from inosine.
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In summary, the industrially promising mutant, S72F/G74D/I153T, was produced by site-directed mutagenesis based on the G74D/I153T mutant, which was generated using the knowledge derived from the random mutagenesis of the homologous enzyme from M.morganii. This suggests that the combined use of site-directed mutagenesis and random mutagenesis is effective and thus amenable to other enzymes. Furthermore, our results demonstrate that rational site-directed mutagenesis, based on an examination of the three-dimensional structure, should be tried even when a substrateenzyme-binding mode is difficult to predict.
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Notes |
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Residues are numbered without considering the 18 residues in the signal peptide, whereas Mihara et al. (Mihara et al., 2000) included signal peptides when numbering the residues of acid phosphatases from other sources
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Acknowledgments |
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References |
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Received November 19, 2001; revised March 8, 2002; accepted March 21, 2002.