Interaction of the two proteins of the methoxylation system involved in cephamycin C biosynthesis. Immunoaffinity, protein cross-linking, and fluorescence spectroscopy studies.
Cephamycin C-producing microorganisms contain a two-protein enzyme system that converts cephalosporins to 7-methoxycephalosporins. Interaction between the two component proteins P7 (Mr 27,000) and P8 (Mr 32,000) has been studied by immunoaffinity chromatography using anti-P7 and anti-P8 antibodies, cross-linking with glutaraldehyde, and fluorescence spectroscopy analysis. Co-renaturation of the P7 and P8 polypeptides resulted in the formation of a protein complex with a molecular mass of 59 kDa, which corresponds to a heterodimer of P7 and P8. Glutaraldehyde cross-linking of the polypeptides after assembly of the protein complex showed the presence of a single heterodimer form that reacted with antibodies against P7 and P8. Each separate protein did not associate with itself into multimers. The P7.P8 complex co-purified by immunoaffinity chromatography from extracts of Nocardia lactamdurans and Streptomyces clavuligerus, suggesting that both proteins are present as an aggregate in vivo. Fluorescence spectroscopy studies of 5-methylaminonaphthalene-1-sulfonyl-P7 in response to increasing concentrations of P8 showed a blue shift in the fluorophore emission, indicating a conformational change of P7 in response to the interaction of P8 with an apparent dissociation constant of 47 microM. NADH showed affinity for the P7 component. The P7.P8 complex interacted strongly with the substrates S-adenosylmethionine and cephalosporin C, differently from that occurring with the separate P7 or P8 components, resulting in a strong blue shift in the fluorescence emission spectra of the complex.
Enguita
F J
FJ
Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, 24071 León, Spain.
Liras
P
P
Leitão
A L
AL
Martín
J F
JF
D002513:Cephamycins / Q000096:biosynthesis*; D002846:Chromatography, Affinity; D003619:Dansyl Compounds / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009615:Nocardia; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D002513:Cephamycins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D009243:NAD; C033853:dansyl acid; C024925:cephamycin C