The crystal structures of chloramphenicol phosphotransferase reveal a novel inactivation mechanism.
Chloramphenicol (Cm), produced by the soil bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyltransferase activity. The Cm-producing streptomycete modifies the primary (C-3) hydroxyl of the antibiotic by a novel Cm-inactivating enzyme, chloramphenicol 3-O-phosphotransferase (CPT). Here we describe the crystal structures of CPT in the absence and presence of bound substrates. The enzyme is dimeric in a sulfate-free solution and tetramerization is induced by ammonium sulfate, the crystallization precipitant. The tetrameric quaternary structure exhibits crystallographic 222 symmetry and has ATP binding pockets located at a crystallographic 2-fold axis. Steric hindrance allows only one ATP to bind per dimer within the tetramer. In addition to active site binding by Cm, an electron-dense feature resembling the enzyme's product is found at the other subunit interface. The structures of CPT suggest that an aspartate acts as a general base to accept a proton from the 3-hydroxyl of Cm, concurrent with nucleophilic attack of the resulting oxyanion on the gamma-phosphate of ATP. Comparison between liganded and substrate-free CPT structures highlights side chain movements of the active site's Arg136 guanidinium group of >9 A upon substrate binding.
Izard
T
T
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK. Tina.Izard@stjude.org
Ellis
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D002701:Chloramphenicol / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D013302:Streptomyces / Q000201:enzymology
D001426:Bacterial Proteins; D011522:Protons; D001224:Aspartic Acid; D002701:Chloramphenicol; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C096858:chloramphenicol 3'-O-phosphotransferase; D008274:Magnesium