Abstract
From analysis of Ramachandran plot for NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH, EC 1.2.1.2), five amino acid residues with non-optimal values φ and ψ have been located in β- and π-turns of the FDH polypeptide chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify their role in the enzyme stability, the residues were replaced with Gly by means of site-directed mutagenesis. The His263Gly mutation caused FDH destabilization and a 1.3-fold increase in the monomolecular inactivation rate constant. The replacements Ala191Gly and Asn234Gly had no significant effect on the stability. The mutations Asn136Gly and Tyr144Gly resulted in higher thermal stability and decreased the inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing effect of the Tyr144Gly mutation was shown to be additive when introduced into the previously obtained mutant FDH with enhanced thermal stability.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
Cantor, C., and Schimmel, P. (1984) Biophysical Chemistry [Russian translation], Mir, Moscow, pp. 248–256.
Brant, D. A., and Schimmel, P. R. (1967) Proc. Natl. Acad. Sci. USA, 58, 428–435.
Gunasekaran, K., Ramakrishnan, C., and Balaram, P. (1996) J. Mol. Biol., 264, 191–198.
Wilmot, C. M., and Thornton, J. M. (1990) Protein Eng., 3, 479–493.
Rajashankar, K. R., and Ramakumar, S. (1996) Protein Sci., 5, 932–946.
Tishkov, V. I., Galkin, A. G., Fedorchuk, V. V., Savitsky, P. A., Rojkova, A. M., Gieren, H., and Kula, M. R. (1999) Biotechnol. Bioeng., 64, 187–193.
Popov, V. O., and Tishkov, V. I. (2003) in Protein Structures: Kaleidoscope of Structural Properties and Functions (Uversky, V. N., ed.) Research Signpost, Kerala, pp. 441–473.
Rojkova, A. M., Galkin, A. G., Kulakova, L. B., Serov, A. E., Savitsky, P. A., Fedorchuk, V. V., and Tishkov, V. I. (1999) FEBS Lett., 445, 183–188.
Lamzin, V. S., Dauter, Z., Popov, V. O., Harutyunyan, E. H., and Wilson, K. S. (1994) J. Mol. Biol., 236, 759–785.
Tishkov, V. I., Matorin, A. D., Rojkova, A. M., Fedorchuk, V. V., Savitsky, P. A., Dementieva, L. A., Lamzin, V. S., Mezentzev, A. V., and Popov, V. O. (1996) FEBS Lett., 390, 104–108.
Tishkov, V. I., Galkin, A. G., Marchenko, G. N., Tsygankov, Y. D., and Egorov, A. M. (1993) Biotechnol. Appl. Biochem., 18, 201–207.
Ramakrishnan, C., and Srinivasan, N. (1990) Curr. Sci., 59, 851–861.
Hutchinson, E. G., and Thornton, J. M. (1994) Protein Sci., 3, 2207–2216.
Tishkov, V. I., and Popov, V. O. (2004) Biochemistry (Moscow), 69, 1252–1267.
Serov, A. E., Rojkova, A. M., and Tishkov, V. I. (2000) Byul. Mosk. Univ., Ser. 2, Khim., 41, 358–361.
Lee, B., and Vasmatzis, G. (1997) Curr. Opin. Biotechnol., 8, 423–428.
Stites, W. E., Meeker, A. K., and Shortle, D. (1994) J. Mol. Biol., 235, 27–32.
Author information
Authors and Affiliations
Corresponding author
Additional information
__________
Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 974–979.
Original Russian Text Copyright © 2005 by Serov, Odintzeva, Uporov, Tishkov.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-188, December 5, 2004.
Rights and permissions
About this article
Cite this article
Serov, A.E., Odintzeva, E.R., Uporov, I.V. et al. Use of Ramachandran Plot for Increasing Thermal Stability of Bacterial Formate Dehydrogenase. Biochemistry (Moscow) 70, 804–808 (2005). https://doi.org/10.1007/s10541-005-0187-z
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/s10541-005-0187-z