Abstract
The oxidation of cephalosporin C (CPC) by D-amino acid oxidase is the first stage in the biocatalytic industrial process of the production of 7-aminocephalosporanic acid, the initial synthon for the production of semisynthetic cephalosporin antibiotics. We determined the kinetic parameters (catalytic constant rate and Michaelis constant) of the wild-type recombinant oxidase from the yeast Trigonopsis variabilis (TvDAAO); three mutant forms of the enzyme with point Met-to-Leu substitutions at positions 104, 156, and 209; and the TvDAAO RDAF quadruple mutant using the dependence of the initial reaction rate on the CPC concentration (the initial rate method). The point substitutions in the mutants are shown to lead primarily to a decrease in the Michaelis constant. The largest (39%) increase in catalytic efficiency kcat/KM is observed for the mutant with the Met156Leu substitution. The Michaelis constant for the increased four times compared to that for the wild-type enzyme in the reaction with CPC, whereas the catalytic efficiency remains almost unchanged due to a four-fold increase in the catalytic rate constant.
Similar content being viewed by others
REFERENCES
Pollegioni, L., Diederichs, K., Molla, G., Umhau, S., Welte, W., Ghisla, S., and Pilone, M.S., J. Mol. Biol., 2002, vol. 324, p. 535. https://doi.org/10.1016/S0022-2836(02)01062-8
Tishkov, V.I. and Khoronenkova, S.V., Biochemistry (Moscow), 2005, vol. 70, no. 1, p. 40. https://doi.org/10.1134/S0006297908130105
Khoronenkova, S.V. and Tishkov, V.I., Biochemistry (Moscow), 2008, vol. 73, p. 1511. https://doi.org/10.1134/S0006297908130105
Pollegioni, L., Molla, G., Sacchi, S., Rosini, E., Verga, R., and Pilone, M.S., Appl. Microbiol. Biotechnol., 2008, vol. 78, p. 1. https://doi.org/10.1007/s00253-007-1282-4
Barber, M.S., Giesecke, U., Reichert, A., and Minas, W., Adv. Biochem. Eng. Biotechnol., 2004, vol. 88, p. 179.
Atroshenko, D.L., Golubev, I.V., Savin, S.S., and Tishkov, V.I., Moscow Univ. Chem. Bull., 2016, vol. 71, no. 4, p. 243. https://doi.org/10.3103/S0027131416040039
Komarova, N.V., Golubev, I.V., Khoronenkova, S.V., and Tishkov, V.I., Russ. Chem. Bull., 2012, vol. 61, no. 7, p. 1489. https://doi.org/10.1007/s11172-012-0193-4
Tishkov, V.I., Cherskova, N.V., Savin, S.S., and Khoronenkova, S.V., RF Patent 2412998, 2009, Byull. Izobret., 2011, no. 6.
Golubev, I.V., Komarova, N.V., Skirgello, O.E., Osipova, T.A., and Tishkov, V.I., Moscow Univ. Chem. Bull., 2014, vol. 69, no. 2, p. 68. https://doi.org/10.3103/S0027131414020035
Funding
This work was carried out with the partial support of the Russian Foundation for Basic Research (project no. 18-34-00594).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare that they have no conflict of interest.
Additional information
Translated by S. Semenova
Abbreviations: CPC, cephalosporin C; DAAO, D-amino acid oxidase; TvDAAO, D-amino acid oxidase from the yeast Trigonopsis variabilis; and 7-ACA, 7-aminocephalosporanic acid
SUPPLEMENTARY MATERIALS
SUPPLEMENTARY MATERIALS
There are no additional materials.
About this article
Cite this article
Atroshenko, D.L., Pometun, A.A., Savin, S.S. et al. Determination of the Kinetic Parameters of a Wild-Type D-Amino Acid Oxidase from Yeast and Its Mutant Forms in a Reaction of Cephalosporin C Oxidation. Moscow Univ. Chem. Bull. 74, 169–172 (2019). https://doi.org/10.3103/S0027131419040023
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.3103/S0027131419040023