The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐n‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐n‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐n‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐n‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
Cytochrome P450 (CYP) heme‐thiolate monooxygenases catalyze the hydroxylation of the C−H bonds of organic molecules. This reaction is initiated by a ferryl‐oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric‐hydroperoxy complex (Cpd 0) and the Fe(III)‐H2O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4‐alkylthiobenzoic acids and 4‐methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol‐pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X‐ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O‐demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O‐demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O‐dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)‐H2O2 complex which would be more abundant in the peroxide‐driven reactions.
The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4‐cyclohexylbenzoic acid but not the aromatic oxidation of 4‐phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4‐benzyl‐, 4‐phenoxy‐ and 4‐benzoyl‐benzoic acid and methoxy‐substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4‐benzylbenzoic acid. The methoxy‐substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4‐(3′‐methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4‐phenylbenzoic acid. 4‐Phenoxy‐ and 4‐benzoyl‐benzoic acid bound with the ether or ketone oxygen atom hydrogen‐bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4‐phenylbenzoic acid; however the F182L mutant was able to catalyse 4‐phenylbenzoic acid oxidation generating 2′‐hydroxy‐, 3′‐hydroxy‐ and 4′‐hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild‐type enzyme, the phenyl ring of 4‐phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.
The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1, that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, Tuberous Sclerosis Complex.We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving metabolic reprogramming and an increased rate of glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through a differential interaction with AMPK.Our findings suggest that unique 'bespoke' combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.
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