CYP116B5: a new class VII catalytically self-sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes.

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N- and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long- and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN- and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.

Electrochemical detection of human cytochrome P450 2A6 inhibition: a step toward reducing dependence on smoking.

Inhibition of human cytochrome P450 2A6 has been demonstrated to play an important role in nicotine metabolism and consequent smoking habits. Here, the "molecular Lego" approach was used to achieve the first reported electrochemical signal of human CYP2A6 and to improve its catalytic efficiency on electrode surfaces. The enzyme was fused at the genetic level to flavodoxin from Desulfovibrio vulgaris (FLD) to create the chimeric CYP2A6-FLD. Electrochemical characterization by cyclic voltammetry shows clearly defined redox transitions of the haem domain in both CYP2A6 and CYP2A6-FLD. Electrocatalysis experiments using coumarin as substrate followed by fluorimetric quantification of the product were performed with immobilized CYP2A6 and CYP2A6-FLD. Comparison of the kinetic parameters showed that coumarin catalysis was carried out with a higher efficiency by the immobilized CYP2A6-FLD, with a calculated kcat value significantly higher (P < 0.005) than that of CYP2A6, whereas the affinity for the substrate (KM) remained unaltered. The chimeric system was also successfully used to demonstrate the inhibition of the electrochemical activity of the immobilized CYP2A6-FLD, toward both coumarin and nicotine substrates, by tranylcypromine, a potent and selective CYP2A6 inhibitor. This work shows that CYP2A6 turnover efficiency is improved when the protein is linked to the FLD redox module, and this strategy can be utilized for the development of new clinically relevant biotechnological approaches suitable for deciphering the metabolic implications of CYP2A6 polymorphism and for the screening of CYP2A6 substrates and inhibitors.

Dynamics and flexibility of human aromatase probed by FTIR and time resolved fluorescence spectroscopy.

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for ß-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.

The oncogenic transcription factor PAX3-FKHR can convert fibroblasts into contractile myotubes.

PAX3-FKHR, the product of a rearrangement of PAX3 with FKHR is the pathogenetic marker for alveolar rhabdomyosarcoma, an aggressive form of childhood cancer. In this work we show that PAX3-FKHR, which is a stronger transcriptional activator relative to PAX3, can lead to two apparently irreconcilable outcomes: transformation and terminal myogenic differentiation. Fibroblasts (10T1/2, NIH3T3, and a newly established murine line named 'Plus') transduced by PAX3-FKHR acquire transformed features such as anchorage independence and loss of contact inhibition and concomitantly undergo various degrees of myogenic conversion depending on the host cells, including, in the case of the Plus line, terminal differentiation into contractile myotubes. This work highlights the potential of PAX3-FKHR to functionally operate as a deregulated Pangene and may have implications with regard to the identity of the precursor cell giving rise to alveolar rhabdomyosarcoma.

Validation of met as a therapeutic target in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue tumor of childhood deriving from skeletal muscle cells. RMS can be classified in two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS), the latter being characterized by the PAX3/7-FKHR translocation. Here we first investigated whether the Met receptor, a transcriptional target of PAX3 and PAX7, has a role in PAX3-FKHR-mediated transformation. Following PAX3-FKHR transduction, Met was up-regulated in mouse embryonal fibroblasts (MEF), NIH 3T3 and C2C12 cells, and they all acquired anchorage independence. This property was lost in low serum but addition of hepatocyte growth factor/scatter factor (HGF/SF) rescued soft-agar growth. Genetic proof that Met is necessary for this PAX3-FKHR-mediated effect was obtained by transducing with PAX3-FKHR MEFs derived from Met mutant (Met(D/D)) and wild-type (Met(+/+)) embryos. Only Met(+/+) MEFs acquired anchorage-independent growth whereas PAX3-FKHR-transduced Met(D/D) cells were unable to form colonies in soft agar. To verify if Met had a role in RMS maintenance, we silenced the receptor by transducing ERMS and ARMS cell lines with an inducible lentivirus expressing an anti-Met short hairpin RNA (shRNA). Met down-regulation significantly affected RMS cells proliferation, survival, invasiveness, and anchorage-independent growth. Finally, induction of the Met-directed shRNA promoted a dramatic reduction of tumor mass in a xenograft model of RMS. Our data show that both ARMS- and ERMS-derived cell lines, in spite of the genetic drift which may have occurred in years of culture, seem to have retained an "addiction" to the Met oncogene and suggest that Met may represent a target of choice to develop novel therapeutic strategies for ARMS.