However, balFd-V and balFd-VII are each located in close vicinity to other putative P450s, as well as FdRs, of unknown function. The putative 7Fe Fd balFd-I, three of the putative [3Fe–4S] Fds balFd-IV, balFd-V and balFd-VII, as well as the presumed [2Fe–2S]-containing Y 27632 balFd-X were selected here for more detailed biochemical studies. Attempts were made to produce
each of the five putative Fds in E. coli as a C-terminal His6-tagged recombinant protein. However, only balFd-V and balFd-VII could be produced efficiently as cofactor-containing proteins. The production of balFd-I and balFd-IV in E. coli Rosetta2(DE3)pLysS yielded colorless His6-tagged recombinant proteins, lacking
the chromophore expected from intact Fe–S clusters. Overexpression of the balFd-X gene in E. coli yielded no recombinant protein with the expected mass. Further studies on these Fds were not pursued here. The production of balFd-V and balFd-VII yielded red-brown-colored holo-proteins that were purified by Ni-NTA and gel filtration chromatography. Each protein eluted selleckchem from a gel filtration column (Superdex 75 10/300 GL, GE Healthcare) with an apparent mass of about 24–26 kDa (O’Keefe et al., 1991). Both proteins were ≥90% homogeneous by SDS-PAGE and analytical reverse-phase HPLC, and yielded ions consistent with the expected masses for the apo-forms by MALDI-MS 6-phosphogluconolactonase (for balFd-V m/z=7826 [M+H]+, calc. 7826.6; for balFd-VII m/z=7897 [M+H]+, calc. 7896.6). The recombinant balFd-V and balFd-VII showed broad UV-Vis absorption maxima at 280–300 nm and 412 nm (Fig. 3), which are typical for oxidized [3Fe–4S] or [4Fe–4S] Fds (Jouanneau et al., 1990; O’Keefe et al., 1991). By comparison, the recombinant His6-tagged [2Fe–2S] spinFd showed the expected absorbance maxima at 275, 328, 420 and 463 nm (Armengaud et al., 2000). Extinction coefficients for balFd-V and balFd-VII were determined using AAS to establish the iron content, with the assumption that one [3Fe–4S] cluster is present
in each polypeptide chain (ɛ412=18 300 M−1 cm−1 for balFd-V and 14 660 M−1 cm−1 for balFd-VII). The values found are close to those reported for two Fds from S. griseolus: Fd-1 (ɛ410=17 000 M−1 cm−1) and Fd-2 (ɛ410=20 100 M−1 cm−1) (O’Keefe et al., 1991). The quantification of acid-labile sulfide was also carried out using a colorimetric assay (Beinert, 1983). Under optimized conditions, the assays yielded 4.01±0.5 and 3.84±0.5 mol S mol−1 protein for balFd-V and balFd-VII, respectively. The OxyB P450 enzymes from the vancomycin biosynthetic gene cluster of A. orientalis (vanOxyB) and from the balhimycin biosynthetic gene cluster of A. balhimycina (balOxyB) were used for CO-binding and activity assays.