Expression and purification of the rifamycin amide synthase, RifF, an enzyme homologous to the prokaryotic arylamine N-acetyltransferases.
The assembly of the polyketide backbone of rifamycin B by the type I rifamycin polyketide synthase, encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. The sequence of the RifF protein from Amycolatopsis mediterranei shows 26% identity and 40% homology with the members of the arylamine N-acetyltransferase (NAT) family of proteins. Based on the homology of the primary structures and the similarity of the reactions catalyzed by the two enzymes, we have compared the RifF protein with members of the NAT family. We have modeled the three-dimensional (3D) structure of RifF using NAT from Salmonella typhimurium and Mycobacterium smegmatis as a template. Proteolytic digestions of RifF revealed accessible regions in the protein which are in agreement with the modeled structure. We have expressed the whole protein and individual domains of the protein based on comparison with NAT from S. typhimurium and have purified the proteins by affinity chromatography using a hexahistidine tag. RifF has been further purified using ion-exchange (Mono Q) chromatography. An antiserum has been generated using the C-terminal nona- and tridecapeptides of RifF and has been shown to recognize RifF uniquely. It does not cross-react with any other member of the NAT family.
Pompeo
Frédérique
F
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Mushtaq
Adeel
A
Sim
Edith
E
D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012294:Rifamycins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D011994:Recombinant Proteins; D012294:Rifamycins; D001191:Arylamine N-Acetyltransferase; D014357:Trypsin