Abstract
A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 → Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 → V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 → P196 mutation that would disrupt a surface α-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
Burleigh, B. D., Rigby, P. W. J., and Hartley, B. S. (1974). Biochem. J. 143, 341-352.
Butler, P. J. G., and Hartley, B. S. (1972). In Methods in Enzymology, Vol. 25 (Hirs, C. H., and Timasheff, S. N., eds:), Academic Press, New York, pp. 191-199.
Cintra, A. C. O., Vieira, C. A., and Giglio, J. R. (1990). J. Protein Chem. 9, 221-227.
Cintra, A. C. O., Marangoni, S., Oliveira, B., and Giglio, J. R. (1993). J. Protein Chem. 12, 57-64.
Dothie, J. M., Giglio, J. R., Moore, C. B., Taylor, S. S., and Hartley, B. S. (1985). Biochem. J. 230, 569-578.
Ghosh, D., Weeks C. M., Grochulski, P., Duax W. L., Erman, M., Rimsay, R. L., and Orr, J. C. (1991). Proc. Natl. Acad. Sci. USA 88, 10064-10068.
Giglio, J. R. (1977). Anal. Biochem. 82, 262-264.
Gray, W. R. (1972). In Methods in Enzymology, Vol. 25, (Hirs, C. H., and Timasheff, S. N., eds.), Academic Press, New York, pp. 333-344.
Guex, N., and Peitsch, M. C. (1997). Electrophoresis 18, 2714-2723.
Hartley, B. S. (1984a). In Microorganisms as Model Systems for Studying Evolution (Mortlock, R. P., ed.), Plenum Press, New York, pp. 23-54.
Hartley, B. S. (1984b). In Microorganisms as Model Systems for Studying Evolution (Mortlock, R. P., ed.), Plenum Press, New York, pp. 55-108.
Hartley, B. S., Altosaar, I., Dothie, J. W., and Neuberger, M. S. (1976). In Structure-Function Relationship of Proteins (Markham, R., and Horn, R. W., eds.), Elsevier/North-Holland, Amsterdam, pp. 191-200.
Hulsmeyer, M., Hecht, H. J., Niefeld, K., Hofer, B., Eltis, L. D., Timmis, K. N., and Schomberg, D. (1998). Protein Sci. 7, 1286-1293.
Itzaki, R. F., and Gill, D. M. (1964). Analyt. Biochem. 9, 401-410.
Laemmli, U. K. (1970). Nature 227, 680-685.
Lerner, S. A., Wu, T. T., and Lin, E. C. C. (1964). Science 146, 1313-1315.
Loviny, T., Norton, P. M., and Hartley, B. S. (1985). Biochem. J. 230, 579-585.
Marangoni, S., Ghiso, J., Sampaio, S. V., Arantes, E. C., Giglio, J. R., Oliveira, B., and Frangione, B. (1990). J. Protein Chem. 9, 595-601.
Mortlock, R. P., Fossit, D. D., and Wood, W. A. (1965). Proc. Natl. Acad. Sci. USA 54, 572-579.
Offord, R. E. (1966). Nature 211, 591-593.
Rigby, W. J., Burleigh, B. D., and Hartley, B. S. (1974). Nature 251, 200-204.
Ryle, A. P., Sanger, F., Smith, L. F., and Kitai, R. (1955). Biochem. J. 60, 541-556.
Skoog, B., and Wichman, A. (1986). Trends Anal. Chem. 5, 82-93.
Taylor, S. S., Rigby, P. W. J., and Hartley, B. S. (1974). Biochem. J. 141, 693-700.
Wu, T. T., Lin, E. C. C., and Tanaka, S. (1968). J. Bact. 96, 447-456.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Homsi-Brandeburgo, M.I., Toyama, M.H., Marangoni, S. et al. The Amino Acid Sequence of Ribitol Dehydrogenase-F, a Mutant Enzyme with Improved Xylitol Dehydrogenase Activity. J Protein Chem 18, 489–495 (1999). https://doi.org/10.1023/A:1020601011846
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1020601011846