[en] The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Rhazi, Noureddine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Charlier, Paulette ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Dehareng, Dominique ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Engher, Danièle
Vermeire, Marcel
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
van Heijenoort, J. (2001) Glycobiology 11, 25R-36R.
Terrak, M., Ghosh, T. K., van Heijenoort, J., Van Beeumen, J., Lampilas, M., Aszodi, J., Ayala, J. A., Ghuysen, J.-M., and Nguyen-Distèche, M. (1999) Mol. Microbiol. 34, 350-364.
Vollmer, W., von Rechenber, M., and Höltje, J. V. (1999) J. Biol. Chem. 274, 6726-6734.
Marrec-Fairley, M., Piette, A., Gallet, X., Brasseur, R., Hara, H., Fraipont, C., Ghuysen, J.-M., and Nguyen-Distèche, M. (2000) Mol. Microbiol. 37, 1019-1031.
Adam, M., Fraipont, C., Rhazi, N., Nguyen-Distèche, M., Lakaye, B., Frère, J.-M., Devreese, B., Van Beeumen, J., van Heijenoort, J., and Ghuysen, J.-M. (1997) J. Bacteriol. 179, 6005-6009.
Höltje, J. V. (1998) Microbiol. Mol. Biol. Rev. 62, 181-203.
Nelson, D. E., and Young, K. D. (2001) J. Bacteriol. 183, 3055-3064.
Frère, J.-M. (1995) Mol. Microbiol. 16, 385-395.
Ghuysen, J.-M., and Dive, G. (1994) New Compr. Biochem. 27, 103-130.
Palomeque-Messia, P., Quittre, V., Leyh-Bouille, M., Nguyen-Distèche, M., Gershater, C. J. L., Dacey, I. K., Dusart, J., Van Beeumen, J., and Ghuysen, J.-M. (1992) Biochem. J. 288, 87-91.
Nguyen-Distèche, M., Leyh-Bouille, M., Pirlot, S., Frère, J.-M., and Ghuysen, J.-M. (1986) Biochem. J. 235, 167-176.
Grandchamps, J., Nguyen-Distèche, M., Damblon, C., Frère, J.-M., and Ghuysen, J.-M. (1995) Biochem. J. 307, 335-339.
Fonzé, E., Vermeire, M., Nguyen-Distèche, M., Brasseur, R., and Charlier, P. (1999) J. Biol. Chem. 274, 21853-21860.
Matagne, A., and Frère, J.-M. (1995) Biochim. Biophys. Acta 1246, 109-127.
Minasov, G., Wang, X., and Shoichet, B. K. (2002) J. Am. Chem. Soc. 124, 5333-5340.
Kelly, J. A., Knox, J. R., Zhao, H., Frère, J.-M., and Ghuysen, J.-M. (1989) J. Mol. Biol. 209, 281-295.
Jamin, M., Wilkin, J. M., and Frère, J.-M. (1995) Essays Biochem. 29, 1-24.
Knox, J. R., Moews, P, C., and Frère, J.-M. (1996) Chem. Biol. 3, 937-947.
Paetzel, M., Danel, F., De Castro, L., Mosimann, S. C., Page, M. G. P., and Strynadka, N. C. J. (2000) Nat. Struct. Biol. 7, 918-925.
Maveyraud, L., Golemi, D., Kotra, L. P., Tranier, S., Vakulenko, S., Mobashery, S., and Samama, J. P. (2000) Struct. Folding Des. 8, 1289-1298.
Delettre, J., Jarlier, V., Collatz, E., and Sougakoff, W. (2001) J. Mol. Biol. 310, 859-884.
Adam, M., Damblon, C., Plaitin, B., Christiaens, L., and Frère, J.-M. (1990) Biochem J. 270, 525-529.
Adam, M., Damblon, C., Jamin, M., Zorzi, W., Dusart, V., Galleni, M., El Kharroubi, A., Piras, G., Spratt, B. G., Keck, W., Coyette, J., Ghuysen, J.-M., Nguyen-Distèche, M., and Frère, J.-M. (1991) Biochem. J. 279, 601-604.
Katz, E., Thompson, C. J., and Hopwood, D. A. (1983) J. Gen. Microbiol. 129, 2703-2714.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-59.
Hopwood, D. A., Kieser, T., Wright, H. M., and Bibb, M. J. (1993) J. Gen. Microbiol. 129, 2257-2269.
Granier, B., Jamin, M., Adam, M., Galleni, M., Lakaye, B., Zorzi, W., Grandchamps, J., Wilkin, J.-M., Fraipont, C., Joris, B., Duez, C., Nguyen-Distèche, M., Coyette, J., Leyh-Bouille, M., Dusart, J., Christiaens, L., Frère, J.-M., and Ghuysen, J.-M. (1994) Methods Enzymol. 244, 249-266.
Jamin, M., Adam, M., Damblon, C., Christiaens, L., and Frère, J.-M. (1991) Biochem. J. 280, 499-506.
Leyh-Bouille, M., Nguyen-Distèche, M., Pirlot, S., Veithen, A., Bourguignon, C., and Ghuysen, J.-M. (1986) Biochem. J. 235, 177-182.
Fraipont, C., Adam, M., Nguyen-Distèche, M., Keck, W., Van Beeumen, J., Ayala, J. A., Granier, B., Hara, H., and Ghuysen, J.-M. (1994) Biochem. J. 298, 189-195.
Siemens Industrial Automation, Inc. (1993) SAINT Software Reference Manual, Madison, WI.
Leslie, A. G. R. W. (1996) MOSFLM Version 5.40 Mosflm Users Guide.
Kabsch, W. (1993) J. Appl. Crystallogr. 26, 795-800.
Brünger, A. T. (1993) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR, Yale University Press, New Haven, CT.
Roussel, A., and Cambillau, C. (1989) Silicon Graphics Geometry Partner Directory, pp 77-78, Silicon Graphics, Mountain View, CA.
Mourey, L., Kotra, L. P., Bellettini, J., Bulychev, A., O'Brien, M., Miller, M. J., Mobasherry, S., and Samama, J.-P. (1999) J. Biol. Chem. 274, 25260-25265.
Jacob, F., Joris, B., Lepage, S., Dusart, J., and Frere, J.-M. (1990) Biochem. J. 277, 399-406.
Jacob, F., Joris, B., Dideberg, O., Dusart, J., Ghuysen, J.-M., and Frere, J.-M. (1990) Protein Eng. 4, 79-86.
Juteau, J.-M., Billings, E., Knox, J. R., and Levesque, R. C. (1992) Protein Eng. 5, 693-701.
Josuna, J., Viadiu, H., Fink, A., and Soberón, X. (1995) J. Biol. Chem. 270, 775-780.
Tsukumoto, K., Tachibana, K., Yamazaki, N., Ishii, Y., Ujiie, K., Nishida, N., and Sawai, T. (1990) Eur. J. Biochem. 188, 15-22.
Monnaie, D., Dubus, A., Cooke, D., Marchand-Brynaert, J., Normark, S., and Frere, J.-M. (1994) Biochemistry 33, 5193-5201.
Beadle, B. M., and Shoichet, B. K. (2002) J. Mol. Biol. 321, 285-296.
Hadonou, A. M., Jamin, M., Adam, M., Joris, B, Dusart, J., Ghuysen, J.-M., and Frere, J.-M. (1992) Biochem. J. 282, 495-500.
Crichlow, C. V., Kuzin, A. P., Nukaga, M., Sawai, T., and Knox, J. R. (1999) Biochemistry 38, 10256-10261.
McDonough, M. A., Anderson, J. W., Silvaggi, N. R., Pratt, R. F., Knox, J. R., and Kelly, J. A. (2002) J. Mol. Biol. 322, 111-122.
Golemi, D., Maveyraud, L., Vakulenko, S., Samama, J.-P., and Mobasherry, S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14280-14285.
Davies, C., White, S. W., and Nicholas, R. A. (2001) J. Biol. Chem. 276, 616-623.
Gordon, E., Mouz, E., Duée, E., and Dideberg, O. (2000) J. Mol. Biol. 299, 477-485.
Sauvage, E., Schoot, B., Marquette, J. P., Leonard, G., Stefanic, P., Kerff, F., Fonzé, E., Taburet, Y., Prevost, D., Dumas, J., Coyette, J., and Charlier, P. (2002) Cell. Mol. Life Sci. 59, 1223-1232.
Lim, D., and Strynadka, N. (2002) Nat. Struct. Biol. 9, 870-876.
Adahi, H., Ishiguro, M., Imajoh, S., Ohta, T., and Matsuzawa, H. (1992) Biochemistry 31, 430-437.
Van der Linden, M. P. G., de Haan, L., Dideberg, O., and Keck, W. (1994) Biochem. J. 303, 357-362.
Chen, C. C., Smith, T. J., Kapadia, G., Wäsch, S., Zawadzeke, L. E., Coulson, A., and Herzberg, O. (1996) Biochemistry 35, 12251-12258.
Graves-Woodward K., and Pratt, R. F. (1998) Biochem. J. 332, 755-761.
Wilkin, J. M., Jamin, M., Joris, B., and Frere, J.-M. (1993) Biochem. J. 293, 195-201.
Fonzé, E., Vanhove, M., Dive, G., Sauvage, E., Frère, J.-M., and Charlier, P. (2002) Biochemistry 41, 1877-1885.