Structure-function studies of two hexapeptide acetyltransferases

Abstract

Described here are the x-ray crystal structures of two xenobiotic acetyltransferases belonging to the hexapeptide acyltransferase superfamily of enzymes. These enzymes displays tandem repeated copies of a six residue 'hexapeptide repeat' sequence motif encoding a Left-Handed parallel beta helix (LOH) structural domain.;The crystal structure of the xenobiotic acetyltransferase from Pseudomonas aeruginosa PA103 (PaXAT) has been determined as well as its complex with the substrate chloramphenicol and the cofactor analog desulpho-Coenzyme A. PaXAT has been found to display low level chloramphenicol acetylation. The active site of trimeric PaXAT is a short tunnel into which chloramphenicol and the cofactor analog desulpho-CoA project from opposite ends. This tunnel is formed by three glycine residues at position i+1 of three adjacent coils of the LbetaH of one subunit and residues from the extended loop and LbetaH of an adjacent subunit. His 79 of the extended loop forms hydrogen bonds from its imidazole NE2 atom to the 3 '-hydroxyl group of chloramphenicol and from its ND1 group to the peptide oxygen of Thr 86.;The crystal structure of streptogramin A acetyltransferase (SatA) from Enterococcus faecium has been determined and refined to 2.5 A resolution. The structures of SatA in complex with virginiamycin M1, a streptogramin A antibiotic and CoA, CoA and acetyl-CoA have also been solved. The active site of the trimeric enzyme is a tunnel formed by two glycine and one alanine residue at position i+1 of three adjacent coils of the LbetaH of one subunit and residues from the extended loop and LbetaH of an adjacent subunit. The substrates bind at opposite ends of this tunnel and the imidazole NE2 of His 82, in an extended loop region of the protein, is in close proximity to the hydroxyl acceptor of the virginiamycin substrate. The interactions of the conserved histidine residues in both SatA and PaXAT with the hydroxyl acceptors of the substrates are similar to those found in the structurally unrelated type III chloramphenicol acetyltransferase. This suggests that these residues may be similarly positioned and tautomerically stabilized to serve as general bases during catalysis.