Structural studies of penicillin acylase

Penicillin acylases are used in the pharmaceutical industry for the prepara tion of antibiotics. The 3-D structure of Penicillin G acylase from Escheri chia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specifici ty for the amide portion of substrate was probed by the structure determina tion of a range of complexes with substitutions around the phenylacetyl rin g of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the stru ctural consequences of mutation at position B71. Protein processing has been analyzed by the construction of site-directed m utants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of a n ES complex with a true substrate, with implications for the enzymatic mec hanism and stereospecificity of the reaction. Mutations that preclude proce ssing have allowed the structure of the precursor, which includes the 54 am ino acid linker region normally removed from between the A and B chains, to be visualized.