Effect of Active Site Pocket Structure Modification of d-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates
d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.
Keywordsd-Stereospecific amidohydrolase Stereospecificity Substrate recognition Hydrophobic pocket
This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 26450124). We thank Dr. T. Bito Faculty of Agriculture, Tottori University, for helpful advice and technical support during this study.
- 10.Arima, J., Shimone, K., Miyatani, K., Tsunehara, Y., Isoda, Y., Hino, T., & Nagano, S. (2016). Crystal structure of d-stereospecific amidohydrolase from Streptomyces sp. 82F2—Insight into the structural factors for substrate specificity. FEBS Journal, 283(2), 337–349. https://doi.org/10.1111/febs.13579.CrossRefGoogle Scholar
- 11.Okazaki, S., Suzuki, A., Komeda, H., Yamaguchi, S., Asano, Y., & Yamane, T. (2007). Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins. Journal of Molecular Biology, 368(1), 79–91. https://doi.org/10.1016/j.jmb.2006.10.070.CrossRefGoogle Scholar
- 13.John, J.. Perona, & Craik, C. S. (1995). Structural basis of substrate specificity in the serine proteases. Protein Science, 4, 337–360.Google Scholar
- 16.Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF Chimera-A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605–1612. https://doi.org/10.1002/jcc.20084.CrossRefGoogle Scholar
- 17.Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W., & Fu, P. C. (1974). Enzymatic determination of total serum cholesterol. Clinical Chemistry, 20(4), 470–475.Google Scholar
- 18.Elyas, Y. Y. A., Miyatani, K., Bito, T., Uraji, M., Hatanaka, T., Shimizu, K., & Arima, J. (2018). Active site pocket of Streptomyces d-stereospecific amidohydrolase has functional roles in aminolysis activity. Journal of Bioscience and Bioengineering. https://doi.org/10.1016/j.jbiosc.2018.03.004.Google Scholar
- 21.Okazaki, S., Suzuki, A., Mizushima, T., Komeda, H., Asano, Y., & Yamane, T. (2008). Structures of D-amino-acid amidase complexed with L-phenylalanine and with L-phenylalanine amide: Insight into the D-stereospecificity of D-amino-acid amidase from Ochrobactrum anthropi SV3. Acta Crystallographica Section D: Biological Crystallography, 64(3), 331–334. https://doi.org/10.1107/S0907444907067479.CrossRefGoogle Scholar
- 22.Andrew, D., & Mesecar, D. E. K. J. (2000). A new model for protein stereospecificity T. Nature, 403, 614–615.Google Scholar