Interaction of the two proteins of the methoxylation system involved in cephamycin C biosynthesis. Immunoaffinity, protein cross-linking, and fluorescence spectroscopy studies.

The Journal of Biological Chemistry
F J EnguitaJ F Martín

Abstract

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 o...Continue Reading

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Jan 15, 2008·Planta·Serena Schmidt von Braun, Enrico Schleiff
May 17, 2011·Biochemistry. Biokhimii︠a︡·M M KutuzovS N Khodyreva
Mar 11, 2006·Journal of Molecular Biology·Linda M OsterInger Andersson
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