An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Phospholipid N-Methyltransferases Produce Various Methylated Phosphatidylethanolamine Derivatives in Thermophilic Bacteria.

One of the most common pathways for the biosynthesis of the phospholipid phosphatidylcholine (PC) in bacteria is the successive 3-fold N-methylation of phosphatidylethanolamine (PE) catalyzed by phospholipid N-methyltransferases (Pmts). Pmts with different activities have been described in a number of mesophilic bacteria. In the present study, we identified and characterized the substrate and product spectra of four Pmts from thermophilic bacteria. Three of these enzymes were purified in an active form. The Pmts from Melghirimyces thermohalophilus, Thermostaphylospora chromogena, and Thermobifida fusca produce monomethyl-PE (MMPE) and dimethyl-PE (DMPE). T. fusca encodes two Pmt candidates, one of which is inactivated by mutation and the other is responsible for the accumulation of large amounts of MMPE. The Pmt enzyme from Rubellimicrobium thermophilum catalyzes all three methylation reactions to synthesize PC. Moreover, we show that PE, previously reported to be absent in R. thermophilum, is in fact produced and serves as a precursor for the methylation pathway. In an alternative route, the strain is able to produce PC by the PC synthase pathway when choline is available. The activity of all purified thermophilic Pmt enzymes was stimulated by anionic lipids, suggesting membrane recruitment of these cytoplasmic proteins via electrostatic interactions. Our study provides novel insights into the functional characteristics of phospholipid N-methyltransferases in a previously unexplored set of thermophilic environmental bacteria. IMPORTANCE In recent years, the presence of phosphatidylcholine (PC) in bacterial membranes has gained increasing attention, partly due to its critical role in the interaction with eukaryotic hosts. PC biosynthesis via a three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid N-methyltransferases (Pmts), has been described in a range of mesophilic bacteria. Here, we expand our knowledge on bacterial PC formation by the identification, purification, and characterization of Pmts from phylogenetically diverse thermophilic bacteria and thereby provide insights into the functional characteristics of Pmt enzymes in thermophilic actinomycetes and proteobacteria.