THE REFINED CRYSTALLOGRAPHIC STRUCTURE OF A DD-PEPTIDASE PENICILLIN-TARGET ENZYME AT 1.6 ANGSTROM RESOLUTION

The D-alanyl-D-alanine peptidase from Streptomyces sp. R61 is a 37,500 dalton exocellular enzyme that has served as a model for membrane-bou nd peptidases that are involved in bacterial cell wall biosynthesis. i nhibition of these enzymes by beta-lactam antibiotics ultimately leads to bacterial cell death. The X-ray crystal structure of the R61 D-ala nyl-D-alanine peptidase has been-solved using multiple isomorphous rep lacement, simulated annealing and-least squares refinement. The space group and unit cell parameters are P2(1)2(1)2(1) With a = 51.1 Angstro m, b = 67.3 Angstrom and c = 102.4 Angstrom. The structure has been re fined using 2 sigma data to 1.6 Angstrom resolution with a crystallogr aphic X-factor of 0.148. The model contains 347 residues (2938 atoms) and 254 solvent molecules. The overall temperature factor is 9.6 Angst rom(2), and the estimated coordinate error is 0.14 Angstrom The protei n consists of a single polypeptide chain organized into two regions. O ne region contains a nine-stranded antiparallel beta-sheet with helice s on both faces; this region includes both the amino and carboxyl term ini. The second region is all helical. Sixty percent of the residues o ccur in helices or beta-sheet. The reactive Ser62 is found between the two regions of the enzyme at the amino end of the protein's longest-h elix which begins with one turn of 3(10) helix and continues with four turns of alpha-helix. The active site is an elongated pocket that con tains four basic and four aromatic residues. An oxyanion hole is forme d by Ser62 NH and Thr301 NH. The pocket also contains the few key resi dues that are conserved in all penicillin-binding proteins and beta-la ctamases. Two of these residues, Lys65 and Tyr159, are among the 16 si de-chains that take on multiple conformations in the R61 crystal struc ture. Three of the 12 proline rings adopt two conformations which we b elieve has not been previously reported. There is no anionic acid equi valent to the catalytic Glu166 found in Class A beta-lactamases. Two o rdered water molecules (O507 and O644) are found buried in the active site and hydrogen-bonded to each other (2.6 Angstrom). O507 could pote ntially act as the hydrolytic water molecule for deacylation. (C) 1995 Academic Press Limited