CRYSTAL-STRUCTURE OF THE ASPARTIC-ACID-199-]ASPARAGINE MUTANT OF CHLORAMPHENICOL ACETYLTRANSFERASE TO 2.35-A RESOLUTION - STRUCTURAL CONSEQUENCES OF DISRUPTION OF A BURIED SALT BRIDGE

The crystal structure of the Asp-199 --> Asn mutant of chloramphenicol acetyltransferase (CAT) has been determined to 2.35-angstrom resolution. In wild-type CAT Asp-199 is involved in a fully buried intrasubunit salt bridge with Arg-18, an interaction that is adjacent to the active site. Replacement of aspartate with asparagine by site-directed mutagenesis disrupts this salt bridge and causes extensive conformational changes within the active site. The imidazole group of the catalytically essential His-195 is reorientated, with the loss of interactions thought to stabilize the preferred tautomer of this residue. Arg-18 and Asn-199 form three new intersubunit interactions as a result of large side-chain torsion angle changes which cause the movement of two polypeptide loops, some residues of which are up to 20-angstrom away from the site of the mutation. The new interactions of Arg-18 and Asn-199 compensate for the loss of the buried salt bridge and afford near-wild-type thermostability to Asn-199 CAT, albeit with a greatly reduced activity.