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.
Highlights
Electron transfer is fundamental for all living organisms, being involved in respiratory processes to produce chemical energy within the cell, pathways to build large molecules from smaller substrates and the breakdown of molecules into smaller species for cellular metabolism
The substrate pro les of several of the cytochrome P450 enzymes can be inferred from sequence homology to those that have been previously studied in other mycobacterial species and inferred from sequence homology.[38,39,40,41,42,43]
The majority of the ferredoxins are of the single cluster type and all have a non-cysteine residue in their iron–sulfur cluster binding motif, but not an alanine or glycine residue that commonly replaces the second cysteine of the motif in [3Fe–4S] ferredoxins
Summary
Electron transfer is fundamental for all living organisms, being involved in respiratory processes to produce chemical energy within the cell, pathways to build large molecules from smaller substrates (anabolic) and the breakdown of molecules into smaller species for cellular metabolism (catabolic). The electron carrier proteins involved in these pathways tightly regulate the shuttling of electrons between the donor and acceptor.[1,2] Monooxygenases are an essential set of enzymes that are intrinsically involved in these anabolic and catabolic processes and require a supply of electrons in order to function. They typically catalyse the selective hydroxylation of a wide range of organic molecules using dioxygen (eqn (1)).[3,4]. These enzymes are a valuable resource for biocatalysis and key targets for antibacterial drug design.[6,7,8]
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