INTERACTION OF THE 2 PROTEINS OF THE METHOXYLATION SYSTEM INVOLVED INCEPHAMYCIN-C BIOSYNTHESIS - IMMUNOAFFINITY, PROTEIN CROSS-LINKING, AND FLUORESCENCE SPECTROSCOPY STUDIES
Fj. Enguita et al., INTERACTION OF THE 2 PROTEINS OF THE METHOXYLATION SYSTEM INVOLVED INCEPHAMYCIN-C BIOSYNTHESIS - IMMUNOAFFINITY, PROTEIN CROSS-LINKING, AND FLUORESCENCE SPECTROSCOPY STUDIES, The Journal of biological chemistry, 271(52), 1996, pp. 33225-33230
Cephamycin C-producing microorganisms contain a two-protein enzyme sys
tem that converts cephalosporins to 7-methoxycephalosporins. interacti
on between the two component proteins P-7 (M(r) 27,000) and P-8 (M(r)
32,000) has been studied by immunoaffinity chromatography using anti-P
-7 and anti-P-8 antibodies, cross-linking with glutaraldehyde, and flu
orescence spectroscopy analysis. Co-renaturation of the P-7 and P-8 po
lypeptides resulted in the formation of a protein complex with a molec
ular mass of 59 kDa, which corresponds to a heterodimer of P-7 and P-8
. Glutaraldehyde cross-linking of the polypeptides after assembly of t
he protein complex showed the presence of a single heterodimer form th
at reacted with antibodies against P-7 and P-8. Each separate protein
did not associate with itself into multimers. The P-7 . P-8 complex co
-purified by immunoaffinity chromatography from extracts of Nocardia l
actamdurans and Streptomyces clavuligerus, suggesting that both protei
ns are present as an aggregate in vivo. Fluorescence spectroscopy stud
ies of 5-methylaminonaphthalene-1-sulfonyl-P-7 in response to increasi
ng concentrations of P-8 showed a blue shift in the fluorophore emissi
on, indicating a conformational change of P-7 in response to the inter
action of P-8 with an apparent dissociation constant of 47 mu M. NADH
showed affinity for the P-7 component. The P-7 . P-8 complex interacte
d strongly with the substrates S-adenosylmethionine and cephalosporin
C, differently from that occurring with the separate P-7 or P-8 compon
ents, resulting in a strong blue shift in the fluorescence emission sp
ectra of the complex.