REPLACEMENT OF CATALYTIC HISTIDINE-195 OF CHLORAMPHENICOL ACETYLTRANSFERASE: EVIDENCE FOR A GENERAL BASE ROLE FOR GLUTAMATE

THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177, TYR 178 AND LEU 187.

Refined Crystal Structure of Type III Chloramphenicol Acetyltransferase at 1.75 Angstroms Resolution

MNYTKFDVKNWVRREHFEFYRHRLPCGFSLTSKIDITTLKKSLDDSAYKFYPVMIYLIAQAVNQFDELRMAIKDDELIVW
DSVDPQFTVFHQETETFSALSCPYSSDIDQFMVNYLSVMERYKSDTKLFPQGVTPENHLNISALPWVNFDSFNLNVANFT
DYFAPIITMAKYQQEGDRLLLPLSVQVHQAVCDGFHVARFINRLQELCNSKLK

SHEET
RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX-STRANDED
BETA SHEET OF AN ADJACENT SUBUNIT OF THE TRIMER, RESULTING
IN A SEVEN-STRANDED SHEET THAT SPANS THE SUBUNIT INTERFACE.

THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177,
TYR 178 AND LEU 187

THE CATALYTICALLY ESSENTIAL HIS 195 (WHICH ACTS AS A GENERAL BASE IN THE PROPOSED CATALYTIC MECHANISM) HAS BEEN MUTATED TO GLUTAMINE. THE RESULTANT ENZYME SHOWS AN ACTIVITY FIVE ORDERS OF MAGNITUDE LOWER THAN WILD TYPE, ALTHOUGH THE KM VALUES FOR BOTH CHLORAMPHENICOL AND ACETYL COA ARE ALMOST UNCHANGED. THE RESIDUAL ACTIVITY DETECTED IS ALMOST CERTAINLY DUE TO MISINCORPORATION OF HISTIDINE AT RESIDUE 195.

THE COBALT IONS PLAY A CRUCIAL ROLE IN STABILIZING THE CRYSTAL LATTICE (LESLIE, 1990).

IN SPITE OF THE FACT THAT CHLORAMPHENICOL WAS PRESENT IN THE CRYSTALLIZATION MOTHER LIQUOR, THERE IS NO EVIDENCE FOR BOUND CHLORAMPHENICOL IN THE STRUCTURE. INSTEAD, A MOLECULE OF 2-MERCAPTOETHANOL (WHICH WAS PRESENT AT 5MM CONCENTRATION) IS FOUND IN THE CHLORAMPHENICOL BINDING POCKET.

THE AMINO ACID NUMBERING SCHEME ADOPTED IS BASED ON THE ALIGNMENT OF SEVERAL CAT SEQUENCES. FOR THE TYPE III ENZYME WHOSE COORDINATES ARE GIVEN HERE, MET 6 IS THE N-TERMINAL RESIDUE AND THERE IS NO RESIDUE 79.

THE STRUCTURE WAS SOLVED BY MOLECULAR REPLACEMENT USING THE REFINED 1.75 ANGSTROMS RESOLUTION STRUCTURE OF THE WILD TYPE ENZYME AS AN INITIAL MODEL.

MNYTKFDVKNWVRREHFEFYRHRLPCGFSLTSKIDITTLKKSLDDSAYKFYPVMIYLIAQAVNQFDELRMAIKDDELIVW
DSVDPQFTVFHQETETFSALSCPYSSDIDQFMVNYLSVMERYKSDTKLFPQGVTPENHLNISALPWVNFDSFNLNVANFT
DYFAPIITMAKYQQEGDRLLLPLSVQVHHAVCDGFHVARFINRLQELCNSKLK