Abstract
Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 → Arg and Leu64 → Gln mutations were essentially inactive and kinetic parameters such as V max and k cat were approximately 1/107 of those of the wild-type enzyme. The Asp98 → Asn mutation did not affect the K m value significantly, but decreased k cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K m nor k cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.
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REFERENCES
Akiyama, K., Fujita, T., Kuroshima, K., Sakane, T., Yokota, A., and
Takata, R. (1999). J. Biosci. Bioeng. 87, 383–385.
Ames, G. F. L. (1974). J. Biol. Chem. 249, 634–644.
Ando, A., Noguchi, K., Yanagi, M., Shinoyama, H., Kagawa, Y., Hirata, H., Yabuki, M., and Fujii, T. (1992). J. Gen. Appl. Microbiol. 38, 135–144.
Boucher, I., Fukamizo, T., Honda, Y., Willick, G. E., Neugebauer, W. A., and Brzezinski, R. (1995). J. Biol. Chem. 270, 31077–31082.
Fukamizo, T. and Brzezinski, R. (1997). Biochem. Cell. Biol. 75, 687–696.
Fukamizo, T., Honda, Y., Toyoda, H., Ouchi, S., and Goto, S. (1996). Biosci. Biotechnol. Biochem. 60, 1705–1708.
Hahn, M., Olsen, O., Politz, O., Borriss, R., and Heinemann, U. (1995). J. Biol. Chem. 270, 3081–3088.
Hocking, J. D. and Harris, J. I. (1976). Experientia Suppl. 26 , 121–133.
Jeuniaux, C. (1966). In Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Academic Press, New York, Vol. 8, pp. 644–650.
Kawata, Y., Tamura, K., Kawamura, M., Ikei, K., Mizobata, T., Nagai, J., Fujita, M., Yano, S., Tokushige, M., and Yumoto, N. (2000). Eur. J. Biochem. 267, 1847–1857.
Kurakake, M., Yo-u, S., Nakagawa, K., Sugihara, M., and Komaki, T. (2000). Curr. Microbiol. 40, 6–9.
Laemmli, U. K. (1970). Nature 227, 680–685.
Lowry, O. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J. (1951). J. Biol. Chem. 193, 265–271.
Marcotte, E. M., Monzingo A. F., Ernst, S. R., Brzezinski, R., and Robertus, J. D. (1996). Nature Struct. Biol. 3, 155–162.
Masson, J. Y., Dennis, F., and Brzezinski, R. (1994). Gene 140, 103–107.
Masson, J. Y., Boucher, I., Neugebauer, W. A., Ramotar, D., and Brzezinski, R. (1995). Microbiology 141, 2629–2635.
Mayer, R. T., McCollum, T. G., Niedz, R. P., Hearn, C. J., McDonald, R. E., Berdis, E., and Doostdar, H. (1996). Planta 200, 289–295.
Miller, L. (1959). Anal. Chem. 31, 426–431.
Pace, C. N., Shirley, B. A., and Thomson, J. A. (1989). In Protein Structure: A Practical Approach (Creighton, T. E., ed.), IRL Press, Oxford, pp. 311–330.
Park, J. K., Shimono, K., Ochiai, N., Shigeru, K., Kurita, M., Ohta, Y., Tanaka, K., Matsuda, H., and Kawamukai, M. (1999). J. Bacteriol. 181, 6642–6649.
Parro, V., Roman, M. S., Galindo, I., Purnelle, B., Bolotin, A., Sorokin, A., and Mellado, R. P. (1997). Microbiology 143, 1321–1326.
Saito, J., Kita, A., Higuchi, Y., Nagata, Y., Ando, A., and Miki, K. (1999). J. Biol. Chem. 274, 30818–30825.
Shimosaka, M., Fukumori, Y., Zhang, X. Y., He, N. J., Kodaira, R., and Okazaki, M. (2000). Appl. Microbiol. Biotechnol. 54, 354–360.
Sun, L., Adams, B., Gurnon, J. R., Ye, Y., and Van Etten, J. L. (1999). Virology 263, 376–387.
Trudel, J. and Asselin, A. (1989). Anal. Biochem. 178, 362–366.
Uchida, Y. and Ohtaka, A. (1989). Meth. Enzymol. 161, 501–505.
Vali, Z., Kilar, F., Lakatos, S., and Venyaminow, S. A. (1980). Biochim. Biophys. Acta 615, 34–47.
Yoon, H. G., Ha, S. C., Lim, Y. H., and Cho, H. Y. (1998). J. Microbiol. Biotechnol. 8, 449–454.
Yoon, H G., Kim, H. Y., Kim, H. K., Kim, K. H., Hwang, H. J., and Cho, H. Y. (1999). J. Microbiol. Biotechnol. 9, 631–636.
Yoon, H. G., Kim, H. Y., Lim, Y. H., Kim, H. K., Shin, D. H., Hong, B. S., and Cho, H. Y. (2000). Appl. Environ. Microbiol. 66, 3727–3734.
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Yoon, H.G., Yang, S.W., Kim, H.Y. et al. Analysis of Essential Leucine Residue for Catalytic Activity of Novel Thermostable Chitosanase by Site-Directed Mutagenesis. J Protein Chem 19, 621–630 (2000). https://doi.org/10.1023/A:1007147214796
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DOI: https://doi.org/10.1023/A:1007147214796