Electron transfer ferredoxins with unusual cluster binding motifs support secondary metabolism in many bacteria

Abstract

The proteins responsible for controlling electron transfer in bacterial secondary metabolism are not always known or characterised. Here we demonstrate that many bacteria contain a set of unfamiliar ferredoxin encoding genes which are associated with those of cytochrome P450 (CYP) monooxygenases and as such are involved in anabolic and catabolic metabolism. The model organism Mycobacterium marinum M contains eleven of these genes which encode [3Fe-4S] or [4Fe-4S] single cluster containing ferredoxins but which have unusual iron-sulfur cluster binding motif sequences, CXX?XXC(X) _n CP, where '?' indicates a variable amino acid residue. Rather than a cysteine residue, which is highly conserved in [4Fe-4S] clusters, or alanine or glycine residues, which are common in [3Fe-4S] ferredoxins, these genes encode at this position histidine, asparagine, tyrosine, serine, threonine or phenylalanine. We have purified, characterised and reconstituted the activity of several of these CYP/electron transfer partner systems and show that all those examined contain a [3Fe-4S] cluster. Furthermore, the ferredoxin used and the identity of the variable motif residue in these proteins affects the functionality of the monooxygenase system and has a significant influence on the redox properties of the ferredoxins. Similar ferredoxin encoding genes were identified across Mycobacterium species, including in the pathogenic M. tuberculosis and M. ulcerans, as well as in a wide range of other bacteria such as Rhodococcus and Streptomyces. In the majority of instances these are associated with CYP genes. These ferredoxin systems are important in controlling electron transfer across bacterial secondary metabolite production processes which include antibiotic and pigment formation among others.

Fig. 1. The X-ray crystal structure of the histidine containing ferredoxins from R. palustris HaA2 (PDB ; 4ID8). (A) Full structure; (B) a zoomed in view of the iron–sulfur cluster with the residues of the ferredoxin binding motif highlighted. The motif of the ferredoxins from M. marinum is highlighted. These residues would replace the histidine which is highlighted in the figure.

Fig. 2. A phylogenetic tree (phenogram) of the [3/4Fe–4S] ferredoxins from M. marinum (Fdx1–Fdx11), M. ulcerans (Mul_1–Mul_7), and M. tuberculosis (Rv0763c, Rv1786 and Rv3503c). The ferredoxins from S. coelicolor, S. lavendulae and the structurally characterised ferredoxins from R. palustris HaA2, P. furiosus, C. thermoaceticum and T. litoralis are included for comparison (see figure). The grouping of the ferredoxins from M. marinum and M. ulcerans show they are closely related (97–100% sequence identity). There is a lower yet significant similarity to the ferredoxins from M. tuberculosis (78–92% sequence identity; note the low 78% value is unusual and arises as the gene Rv3503c is shorter than Fdx11 by the equivalent of nineteen amino acids). For the majority of the ferredoxins there is a low similarity to those from other bacterial species, for example Fdx1 has only 35% sequence identity with the structurally characterised R. palustris HaA2 ferredoxin (PDB: ; 4ID8). The threonine containing Fdx2 has the closest relationship with the [3Fe–4S] ferredoxins from Streptomyces species while the [4Fe–4S] ferredoxins from the thermophiles P. furiosus, C. thermoaceticum and T. litoralis cluster together.,,–

Fig. 3. CYP147G1 oxidation of undecanoic acid to 10-hydroxyundecanoic acid by CYP147G1 and the oxidation of β-ionone to 4-hydroxy-β-ionone by CYP278A1 and CYP150A5.

Fig. 4. CYP147G1 product formation is reduced when supported by the mutant Fdx partners. (A) GC-MS chromatogram of the CYP147G1 turnover of undecanoic acid (black trace) after derivatisation with BSTFA/TMSCl. Derivatised undecanoic acid (RT 9.2 min, control red trace) and the 10-hydroxyundecanoic acid (RT 13.9 min) are shown. The chromatogram has been offset along the x and y axes for clarity. (B) Quantitation of the 10-hydroxyundecanoic acid product from variant Fdx3/CYP147G1 whole-cell turnovers of undecanoic acid. The axis shows the triplicate average of the area of integrated product peak divided by the area of the internal standard peak. Error bars show one standard deviation.

Fig. 5. The UV/Vis absorbance spectra of aerobically purified Fdx4 (Fdx3973, black) and Fdx8 (Fdx4736, red) from M. marinum. Other spectra are included in the ESI.

Fig. 6. CD monitored potentiometric titrations of Fdx4 (Mmar3973, panel A), Fdx5 (Mmar4716, panel B) and Fdx9 (Mmar4763, panel C). Traces taken as representing fully oxidized and, where accessible, fully reduced cluster are shown in bold (for Fdx9 the bold traces correspond to the highest and lowest potentials at which spectra were recorded). Red arrows in panels A and B indicate the direction of spectral change upon reduction of the [3Fe–4S] cluster. Panel D shows the relative proportion of oxidized cluster for Fdx4 (black) and Fdx5 (blue) as a function of potential. Solid line shows the predicted behaviour for a single electron oxidation/reduction event with a midpoint potential of +120 mV vs. SHE (black line) or + 230 mV vs. SHE (blue line).