EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5)

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix. © 2022 The Author

CYP116B5hd, a self-sufficient P450 cytochrome: A dataset of its electronic and geometrical properties

This paper documents the dataset obtained from the Electron Paramagnetic Resonance (EPR) study of the electronic properties of a self-sufficient cytochrome P450, CYP116B5hd, which possesses an interesting catalytic activity for synthetic purposes. In fact, when isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Raw data shown in Famulari et al. (2022) and supplementary data in raw and processed forms (figures) are documented and available in this paper. Additionally, simulations of the experimental data together with simulation scripts based for EasySpin, a widespread MATLAB toolbox for EPR spectral simulations, are provided. The procedure for g-value analysis based on a crystal-field theory is also detailed here, offering an interesting tool for comparison of Fe(III)-heme P450 systems. Due to the catalytic interest of the protein, which has been recently discovered, and the correlation that has been reported between g-values and peroxidase function, both, CW-EPR and HYSCORE spectra and data set of the model CYPBM3hd are also provided. Finally, the materials and methods for enzyme production and purification, sample preparation and experimental and spectroscopic procedures a together with instrumental details are described in detail. The data files and simulation scripts can be found in: https://doi.org/10.5281/zenodo.641862

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density

Selective recognition of the di/trimethylammonium motif by an artificial carboxycalixarene receptor

Chemical tools that recognise post-translational modifications have promising applications in biochemistry and in therapy. We report a simple carboxycalixarene that selectively binds molecules containing di/trimethylammonium moieties in isolation, in cell lysates and when incorporated in histone peptides. Our findings reveal the potential of using carboxycalixarene-based receptors to study epigenetic regulation