Search

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H 2 O 2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H 2 O 2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H 2 O 2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H 2 O 2 will enable new structural biology methods and techniques to be applied to study their mechanism of action., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Norcarane is a mechanistic probe of monooxygenase enzymes that is able to detect the presence of cationic or radical intermediates. The addition of substituents around the bicycloheptane ring of the norcarane scaffold can assist in improving enzyme binding affinity and thus improve the regioselectivity of oxidation. Here we prepare in three-step, diastereoselective syntheses, ten norcaranes monosubstituted α to the cyclopropane as advanced probes. Four of these compounds were examined in enzyme binding experiments to evaluate their potential as probe substrates. Additionally, 19 potential products of enzymatic oxidation were generated via two additional synthetic steps for use as product standards in future studies.

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.

The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efficient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with different meta-substituted benzoic acids in order to establish which criteria are important for efficient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efficiently oxidized. For example, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modified position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the CH bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These findings were confirmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efficient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic effect on the activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).

The evolution of CYP102A1 variants with enhanced activity and altered specificity characteristics.

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.