D-alanine:D-alanine ligase, cephalosporin $\beta$-lactamase, and complexes of new $\beta$-lactams with their target enzyme: X-ray structural studies

Date of Completion

January 1990

Keywords

Chemistry, Pharmaceutical|Biophysics, General

Degree

Ph.D.

Abstract

A bacterial cell-wall synthetic enzyme, D-alanine:D-alanine ligase from Salmonella typhimurium, was crystallized in a tetragonal lattice from 12% methoxypolyethylene glycol 5K, 50 mM Hepes, 10 mM D-cycloserine, pH 7.0. The single crystals measured 0.3 mm $\times$ 0.3 mm $\times$ 0.4 mm and could diffract X-rays to 2.3 A resolution.^ Complexes of the D-alanyl-D-alanine peptidase of Streptomyces R61 with antibiotics cephalothin and cefotaxime were crystallographically studied at 2.25 A. Both cephalosporins are observed to be acylated by the catalytic serine 62 of the target enzyme in the complexes. The methoxy group of the acylamido side chain of cefotaxime is buried, and its aminothiazole is more toward the solvent than the thiophene of cephalothin. The overall structures of the two bound cephalosporins are about 1.0 A more exposed to solvent than is the bound cephalosporin C. The larger opening between cephalothin or cefotaxime and the interior surface of the catalytic site may weaken the nonbonded interactions with the enzyme, may also allow more water molecules to enter for deacylation of the ester bonds, so that the half-lives of their complexes with the enzyme are shorter than that of the complex of cephalosporin C and the enzyme.^ A cephalosporinase from Enterobacter cloacae P99 was subjected to X-ray structural determination. Based on one K$\sb3$UO$\sb2$F$\sb5$ derivative, a 5.5 A electron density map calculated in space group P2$\sb1$2$\sb1$2 by iterative single isomorphous replacement (ISIR) phasing shows reasonable and distinguishable molecular boundaries. The density was globally fitted with the known structure of the smaller Class A $\beta$-lactamase from Bacillus licheniformis, which was also used as a model for the molecular replacement searches. There is 10$\sp\circ$ difference in spatial orientation between the ISIR-fit model and the model obtained from the molecular replacement searches. Aligned with either orientation, a P99 model structure was refined with simulated annealing for one cycle. For all 2.3 A resolution data, the final crystallographic R factor was 31.2%. The preliminarily-refined structure indicates the cephalosporinase is closer to the DD-peptidase in three-dimensional folding than to the Class A $\beta$-lactamase. ^

Share

COinS