PURIFICATION AND CRYSTALLOGRAPHIC STUDIES OF A D-ALANYL-D-ALANINE CARBOXY-PEPTIDASE FROM B. STEAROTHERMOPHILUS. Alexandre P. Kuzin¹, Janet L. Frost², Steven L. Condron¹, and Judith A. Kelly¹, ¹Department of Molecular and Cell Biology and Institute of Materials Science, University of Connecticut, Storrs, CT 06269-3125, ²Department of Science, Capital Community Technical College, Hartford, CT 06105

D-alanyl-D-alanine carboxypeptidases (Cpases) are involved in bacterial cell wall biosynthesis and are targets of (-lactam antibiotics. The gene for a Cpase from B. stearothermolphilus has been modified by R. Manning and C. Despreaux of Hoffmann-LaRoche to remove a 26-residue C terminal fragment that anchors the enzyme in the bacterial membrane. The engineered gene was inserted in Pichia pastoris, and the soluble 43kDa enzyme was excreted into the medium. Ammonium sulfate precipitation and Fast Protein Liquid Chromatography using a Mono-S (cationic) column were used to purify the enzyme. The enzyme can also be solubilized via trypsin or chymotrypsin digestion. Cpase was crystallized from 20mM Tris buffer, pH 7.5, with 0.2M NaCl and 10% PEG 8000 in P2₁2₁2 (a=85.1Å, b=140.3Å, c=167.3Å). There are at least four molecules in the asymmetric unit cell. Native data to 3.2Å and data for a K₂PtCl₄ derivative have been collected. The sequence of Cpase was compared to that of the DD-transpeptidase from Streptomyces K15 (Englebert, S. et al., 1994, J. Mol. Biol. 241, 295-297) and aligned using the Multiple Sequence Alignment program (Gupta, S. K. et al., J. Comput. Biol., in press). Seventy of the 262 residues in K15 were found to be identical to the Cpase. Efforts are under way to phase the Cpase with molecular replacement using X-PLOR and AMoRe with K15 as the search model.

This research is sponsored in part by the Connecticut Space Grant Consortium. Assistance of Fritz Winkler of Hoffmann-LaRoche, Basel, and Ron Manning and Carl Despreaux of Hoffmann-LaRoche, Nutley, is gratefully acknowledged.