Bacterial monooxygenases are vital to biogeochemical cycles and are useful for bioremediation and biocatalysis. The soluble di-iron monooxygenases (SDIMOs) are of special interest due to their broad substrate range, high enantioselectivity, and ability to oxidise very inert substrates (e.g. methane). Six groups of SDIMOs were originally defined based on sequence analysis, but the explosion of genomic and metagenomic sequences in recent years has led to new SDIMOs being identified which cannot easily be classified, and have unknown functions and significance.
The draft genome of Solimonas soli, a soil Gammaproteobacteria, contains an unusual SDIMO first identified by Osborne and Haritos (Mol.Phyl.Evol. 2019). The S. soli SDIMO clusters near the root of the SDIMO phylogenetic tree, and is distinct from the two established major radiations of aromatic versus aliphatic SDIMOs. In the present study, we characterised this novel SDIMO in more detail, with the aims of clarifying its phylogenetic position, elucidating its substrates, and predicting its significance.
We detected 21 uncharacterised SDIMOs in Proteobacteria genomes with high similarity to the putative S. soli enzyme (>60% aa identity in the alpha hydroxylase subunit). Based on sequence identity, gene cluster organisation, amino acid motifs, and bootstrap support, we propose that the S. soli SDIMO and its relatives comprise a new group of SDIMOs i.e. “group 7”. The S. soli SDIMO was cloned into the broad host range vector pUS250 to test its activity. No monooxygenase activity was detected in Escherichia coli, but after transfer into Pseudomonas putida KT2440, oxidation of ethene to epoxyethane was seen, and an apparent specific activity of 0.45 nmol/min/mg protein was calculated.
We have established a coherent new group 7 SDIMO clade, and confirmed that at least one member of this family has alkene oxidation activity. This is the first evidence for the biochemical activity of any member of this SDIMO group. Experiments are in progress using both the native host and the recombinant KT2440 clone to elucidate the substrate range of the group 7 SDIMOs, and predict their ecological significance and possible applications.