Characterization of a virally encoded flavodoxin that can drive bacterial cytochrome P450 monooxygenase activity

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Lamb, D. C., Goldstone, J. V., Zhao, B., Lei, L., Mullins, J. G. L., Allen, M. J., Kelly, S. L., & Stegeman, J. J. Characterization of a virally encoded flavodoxin that can drive bacterial cytochrome P450 monooxygenase activity. Biomolecules, 12(8), (2022): 1107, https://doi.org/10.3390/biom12081107.Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.The study was supported by the National Institutes of Health grant 5U41HG003345 (J.V.G.), by the Woods Hole Center for Oceans and Human Health, NIH P01 ES021923 and NSF OCE-1314642 (J.J.S.), and by a Fulbright Scholarship (to D.C.L.). Funding at Swansea University supported by the European Regional Development Fund/Welsh European Funding Office via the BEACON project (S.L.K)