Hydroxylation Of Camphor Research Articles

Hydroxylation Regiochemistry Is Robust to Active Site Mutations in Cytochrome P450cam (CYP101A1).

Cytochrome P450cam (CYP101A1) catalyzes the hydroxylation of d-camphor by molecular oxygen. The enzyme-catalyzed hydroxylation exhibits a high degree of regioselectivity and stereoselectivity, with a single major product, d-5-exo-hydroxycamphor, suggesting that the substrate is oriented to facilitate this specificity. In previous work, we used an elastic network model and perturbation response scanning to show that normal deformation modes of the enzyme structure are highly responsive not only to the presence of a substrate but also to the substrate orientation. This work examines the effects of mutations near the active site on substrate localization and orientation. The investigated mutations were designed to promote a change in substrate orientation and/or location that might give rise to different hydroxylation products, while maintaining the same carbon and oxygen atom balances as in the wild type (WT) enzyme. Computational experiments and parallel in vitro site-directed mutations of CYP101A1 were used to examine reaction products and enzyme activity. 1H-15N TROSY-HSQC correlation maps were used to compare the computational results with detectable perturbations in the enzyme structure and dynamics. We found that all of the mutant enzymes retained the same regio- and stereospecificity of hydroxylation as the WT enzyme, with varying degrees of efficiency, which suggests that large portions of the enzyme have been subjected to evolutionary pressure to arrive at the appropriate sequence-structure combination for efficient 5-exo hydroxylation of camphor.

Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450cam (CYP101A1)

The camphor-degrading microorganism, Pseudomonas putida strain ATCC 17453, is an aerobic, gram-negative soil bacterium that uses camphor as its sole carbon and energy source. The genes responsible for the catabolic degradation of camphor are encoded on the extra-chromosomal CAM plasmid. A monooxygenase, cytochrome P450cam, mediates hydroxylation of camphor to 5-exo-hydroxycamphor as the first and committed step in the camphor degradation pathway, requiring a dioxygen molecule (O2) from air. Under low O2 levels, P450cam catalyzes the production of borneol via an unusual reduction reaction. We have previously shown that borneol downregulates the expression of P450cam. To understand the function of P450cam and the consequences of down-regulation by borneol under low O2 conditions, we have studied chemotaxis of camphor induced and non-induced P. putida strain ATCC 17453. We have tested camphor, borneol, oxidized camphor metabolites and known bacterial attractants (d)-glucose, (d) - and (l)-glutamic acid for their elicitation chemotactic behavior. In addition, we have used 1-phenylimidazole, a P450cam inhibitor, to investigate if P450cam plays a role in the chemotactic ability of P. putida in the presence of camphor. We found that camphor, a chemoattractant, became toxic and chemorepellent when P450cam was inhibited. We have also evaluated the effect of borneol on chemotaxis and found that the bacteria chemotaxed away from camphor in the presence of borneol. This is the first report of the chemotactic behaviour of P. putida ATCC 17453 and the essential role of P450cam in this process.

Stereo-preference of camphor for H-bonding with phenol, methanol and chloroform: A combined matrix isolation IR spectroscopic and quantum chemical investigation.

Camphor is known to be held in the substrate pocket of cytochrome P450cam enzyme via H-bond with a tyrosine residue of the enzyme in a unique orientation. This structural exclusivity results in regio- and stereo-specific hydroxylation of camphor by the enzyme. We have carried out a combined IR spectroscopic and quantum chemical investigation to shed light on the factors influencing the conformational exclusivity of 1R-(+)-camphor in the substrate pocket of Cytochrome P450cam, and to determine whether the selectivity is an inherent property of the substrate itself, or is imposed by the enzyme. For this purpose, complexes of camphor have been studied with three H-bond donors namely phenol, methanol and chloroform. Each of the three donors was found to form stable complexes with two distinct conformers; the one mimicking the conformation in enzyme substrate pocket was found to be more stable of the two, for all three donors. Experimentally, both conformers of the H-bonded complexes were identified separately for phenol and methanol in an argon matrix at 8 K, but not for chloroform due to very small energy barrier for interconversion of the two conformers. In room temperature solution phase spectra of camphor with all three donors, the differences in spectral attributes between the two isomeric H-bonded complexes were lost due to thermal motions.

Hydrogen Abstraction of Camphor Catalyzed by Cytochrome P450cam: A QM/MM Study.

A combined quantum mechanics and molecular mechanics (QM/MM, QM = UB3LYP-D3, MM = AMBER) method is used to study the hydrogen abstraction reaction in P450cam catalyzed hydroxylation of camphor in the quartet state. Compared to QM/MM calculations in the literature, this study uses larger basis sets for the most important atoms at the active site and QM/MM Hessian harmonic frequency calculations to determine the standard Gibbs free energy of activation and kinetic isotope effect. The QM/MM covalent boundary is treated with a capping hydrogen atom method, which is simple and robust. An energy barrier of 21.3 kcal/mol and a standard free energy of activation of 16.8 kcal/mol are obtained for this hydrogen abstraction reaction. These values are similar to those reported in the literature, suggesting that when a general protocol is followed, QM/MM results are reproducible. It is found that using a sufficiently large basis set is important to minimize basis set errors.

In vivo and in vitro hydroxylation of cineole and camphor by cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

Cytochromes P450 (P450s) are valuable enzymes that can generate a range of useful compounds via biocatalytic oxidations that complement traditional synthetic chemistry. In this study three bacterial P450s, P450cam (CYP101A1), CYP101B1 and the mutant N242A-P450cin (N242A-CYP176A1), were used to produce a range of products from the oxidation of the monoterpenes (1R)- and (1S)-camphor and 1,8-cineole. We demonstrate that both in vitro and in vivo catalytic turnover with these P450s can produce a complement of up to seven hydroxycamphors and seven hydroxycineoles, in addition to compounds produced from further oxidation. The CYP101B1 whole cell catalytic system was found to produce 300–600mg/L of culture of oxidation products that could be easily separated chromatographically. The CYP101B1 in vitro oxidation of 1,8-cineole primarily produced (1S)-5α-hydroxycineole, which was 78% of the total product formed. However, the amount of (1S)-5α-hydroxycineole was reduced to 42% of the total products when isolated from the CYP101B1 whole cell system. (1S)-6α-Hydroxycineole (96% ee) could be isolated from a whole cell catalytic turnover of 1,8-cineole by N242A-P450cin in a yield of 46mg/L (98% of the total product). However, the amount of product isolated ((1R)-5-endo-hydroxycamphor, 75% of the total products) from the whole cell catalytic oxidation of (1R)-camphor with N242A-P450cin was much lower (6mg/L) due to the inefficient use of reducing equivalents (3.5±0.5%) for substrate oxidation. These compounds will assist in the identification of specific structures in mechanistic investigations and structure elucidation, but further optimisation is required to generate larger quantities for synthetic applications.

Successful application of the DBLOC method to the hydroxylation of camphor by cytochrome p450.

The activation barrier for the hydroxylation of camphor by cytochrome P450 was computed using a mixed quantum mechanics/molecular mechanics (QM/MM) model of the full protein-ligand system and a fully QM calculation using a cluster model of the active site at the B3LYP/LACVP*/LACV3P** level of theory, which consisted of B3LYP/LACV3P** single point energies computed at B3LYP/LACVP* optimized geometries. From the QM/MM calculation, a barrier height of 17.5 kcal/mol was obtained, while the experimental value was known to be less than or equal to 10 kcal/mol. This process was repeated using the D3 correction for hybrid DFT in order to investigate whether the inadequate treatment of dispersion interaction was responsible for the overestimation of the barrier. While the D3 correction does reduce the computed barrier to 13.3 kcal/mol, it was still in disagreement with experiment. After application of a series of transition metal optimized localized orbital corrections (DBLOC) and without any refitting of parameters, the barrier was further reduced to 10.0 kcal/mol, which was consistent with the experimental results. The DBLOC method to CH bond activation in methane monooxygenase (MMO) was also applied, as a second, independent test. The barrier in MMO was known, by experiment, to be 15.4 kcal/mol. After application of the DBLOC corrections to the MMO barrier compute by B3LYP, in a previous study, and accounting for dispersion with Grimme's D3 method, the unsigned deviation from experiment was improved from 3.2 to 2.3 kcal/mol. These results suggested that the combination of dispersion plus localized orbital corrections could yield significant quantitative improvements in modeling the catalytic chemistry of transition-metal containing enzymes, within the limitations of the statistical errors of the model, which appear to be on the order of approximately 2 kcal/mole.

Inside Back Cover: Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450cam‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide (Chem. Eur. J. 43/2015)

Inside Back Cover: Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450_cam‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide (Chem. Eur. J. 43/2015)

Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450cam -Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide.

The hydroperoxo iron(III) intermediate P450cam Fe(III) -OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450cam , could be transiently observed in the peroxo-shunt oxidation of the substrate-free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450cam Fe(III) -OOH species as a function of varying reaction conditions, such as pH, and concentration of H2 O2 , camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate-free form of P450cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid-base catalyst and is mainly governed by the ability of H2 O2 to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$. Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic OO bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)-camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe(III) -OOH intermediate in the oxidation of the natural substrate of P450cam .

Monoterpene hydroxylation with an artificial self-sufficient P450 utilizing a P450SMO reductase domain for the electron transfer

Cytochrome P450SMO from Rhodococcus sp. ECU0066 is a natural self-sufficient P450 monooxygenase, consisting of a heme domain, a flavin-reductase domain containing FMN and NADPH binding sites, and a [Fe2S2] ferredoxin domain. P450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor. The variant P450cam (Y96F/V247L) was reported for the oxidation of monoterpene by protein engineering. In this work, we constructed an artificial self-sufficient P450-type monoterpene hydroxylase by connecting the P450SMO reductase domain and the P450cam (Y96F/V247L) domain together with a linker region (G4S)4. The resultant chimeric P450 enzyme could catalyze the hydroxylation of (−)-limonene and α-pinene as well as camphor, which were all inactive for the natural fusion protein P450SMO. Co-expression of the fused P450 with a glucose dehydrogenase (GDH) improved the (−)-limonene conversion as sufficient NADPH was regenerated in the system with glucose as a cosubstrate. This work illustrated that P450SMO reductase might act as an electron donor partner of P450s and might be used for fusion with heterogeneous P450 domains to elucidate the catalytic function of other unknown P450s.

Molecular dynamics of the P450cam-Pdx complex reveals complex stability and novel interface contacts.

Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5-exo-hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe2 S2 containing protein. Two recent crystal structures of the P450cam-Pdx complex, one solved with the aid of covalent cross-linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross-linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross-linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251-Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251-Arg186 ion pair may be important.

Synergistic effects of mutations in cytochrome P450cam designed to mimic CYP101D1.

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.

The Use of Deuterated Camphor as a Substrate in 1H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OH(exo) and C5-H(endo)] from camphor. When D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, the magnitude of the C5-OH(exo) signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.

Water as biocatalyst in cytochrome P450

According to previous quantum mechanics/molecular mechanics (QM/MM) studies, camphor hydroxylation in cytochrome P450 is catalysed by a single water molecule which lowers the computed B3LYP/CHARMM barrier by about 4 kcal mol(-1). Gas-phase B3LYP model studies for a variety of different substrates show the generality of this effect. Its origin is an electrostatic enhancement of hydrogen bonding in the transition state for hydrogen abstraction. Attempts are made to correlate the slight variations in the calculated barrier lowerings with substrate properties. Individual water molecules also have a decisive influence on other reactions in cytochrome P450cam, for instance, on the relative propensity for coupling and uncoupling upon protonation of Compound 0 in the wild-type enzyme and its mutants. These and other examples are reviewed briefly. Finally, we address some methodological issues on how to handle the possible involvement of water molecules in biocatalysis at the QM/MM level.

Inclusion of Dispersion Effects Significantly Improves Accuracy of Calculated Reaction Barriers for Cytochrome P450 Catalyzed Reactions

Prediction of cytochrome P450 reactivity is of great importance to the development of new medicinal compounds. Density functional theory (DFT) has proven itself as a useful tool in the characterization of the elusive reactive species, compound I, and of the mechanisms of substrate oxidation. B3LYP is the most widely used density functional in the study of P450s; however, a major drawback of B3LYP is its inaccurate treatment of dispersion, leading to discrepancies between experiment and theory in some systems. Recent work has shown that an added empirical dispersion correction to B3LYP (B3LYP-D) yields more promising results for similar systems. In the present work, two previously studied systems, camphor hydroxylation and alkene oxidation, have been recalculated using B3LYP-D. Our work shows that inclusion of dispersion has a significant effect on the energies and geometries of transition states and encounter complexes; furthermore, an improved agreement with experimental data is observed.

The Rate-Limiting Step in P450 Hydroxylation of Hydrocarbons A Direct Comparison of the “Somersault” versus the “Consensus” Mechanism Involving Compound I

Model theoretical quantum mechanical (QM) calculations are described for the P-450 hydroxylation of methane, isobutane, and camphor that compare the concerted somersault H-abstraction mechanism with the oxidation step involving Cpd I. Special emphasis has been placed on maintaining a balanced basis set in the oxidation step. QM calculations, employing the 6-311+G(d,p) basis set on the Fe atom and all of the key surrounding atoms involved in the C-H abstraction step, reaffirm a mechanism involving rearrangement of the iron hydroperoxide group (FeO-OH --> FeO...HO(*)) in concert with hydrogen abstraction from the C-H bond of the substrate by the incipient bound hydroxyl radical HO(*). The barrier for the somersault rearrangement of model Cpd 0 (FeO-OH) is calculated to be 21.4 kcal/mol in the absence of substrate. The overall activation energy for the oxidation of camphor involving the somersault motion of the FeO-OH group of P450 model porphyrin iron(III) hydroperoxide [Por(SH)Fe(III)-OOH(-)] --> [Por(SH)Fe(III)-O....HO(-)] in concert with hydrogen abstraction is DeltaE(++) = 12.4 kcal/mol. The corresponding abstraction of the hydrogen atom from the C-H bond of camphor by Cpd I has an activation barrier of 17.6 kcal/mol. Arguments are presented that the somersault rearrangement is induced by steric compression at the active site. Kinetic isotope effect data are discussed that provides compelling evidence for a rate-limiting step involving C-H bond cleavage.

Production and Characterization of a Functional Putidaredoxin Reductase−Putidaredoxin Covalent Complex

In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on Pdx were activated. The yield of the EDC-mediated cross-link depended on the Pdx variant used and the redox state of both partners, decreasing in the following order: Pdr(ox)-Pdx(ox) > Pdr(ox)-Pdx(red) > or = Pdr(red)-Pdx(red). The Pdr-Pdx C73S/C85S conjugate was purified and characterized. Compared to the equimolar mixture of intact Pdr and Pdx, the fusion protein was more efficient in electron transfer to cytochrome c and, in the presence of saturating levels of P450cam, more effectively supported camphor hydroxylation. On the basis of our results, we conclude that (i) the cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, (ii) molecular recognition between Pdr and Pdx is redox-controlled and assisted by the Glu72(Pdx)-Lys409(Pdr) charge-charge interactions, and (iii) the high specificity of the Pdr-Pdx couple may be due to finely tuned interactions at the protein-protein interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.

Fundamental Differences of Substrate Hydroxylation by High-Valent Iron(IV)-Oxo Models of Cytochrome P450

An Iron(IV)-oxo heme(+*) complex (Compound I, Cpd I) is the proposed active species of heme enzymes such as the cytochromes P450 and is elusive; therefore, biomimetic studies on active site mimics give valuable insight into the fundamental properties of heme active species. In this work we present density functional theory (DFT) calculations on substrate hydroxylation by a Compound I mimic [Fe(IV)=O(Por(+*))Cl] and its one-electron reduced form [Fe(IV)=O(Por)Cl](-). Thus, recent experimental studies showed that [Fe(IV)=O(Por)Cl](-) is able to react with substrates via hydride transfer reactions [Jeong, Y. J.; Kang, Y.; Han, A.-R.; Lee, Y.-M.; Kotani, H.; Fukuzumi, S.; Nam, W. Angew. Chem., Int. Ed. 2008, 47, 7321-7324]. By contrast, theoretical studies on camphor hydroxylation by these two oxidants concluded that the one-electron reduced form of Compound I is a sluggish oxidant of hydroxylation reactions [Altun, A.; Shaik, S.; Thiel, W. J. Am. Chem. Soc. 2007, 129, 8978-8987]. To resolve the question why the one-electron reduced Compound I is an oxidant in one case and a sluggish oxidant in other cases, we have performed a DFT study on 10-methyl-9,10-dihydro acridine (AcrH(2)) hydroxylation by [Fe(IV)=O(Por(+*))Cl] and [Fe(IV)=O(Por)Cl](-). The calculations presented in this work show that both [Fe(IV)=O(Por(+*))Cl] and [Fe(IV)=O(Por)Cl](-) are plausible oxidants, but [Fe(IV)=O(Por(+*))Cl] reacts via much lower reaction barriers. Moreover, [Fe(IV)=O(Por(+*))Cl] reacts via hydride transfer, while [Fe(IV)=O(Por)Cl](-) by hydrogen abstraction. The differences between hydride and hydrogen atom transfer reactions have been rationalized with thermodynamic cycles and shown to be the result of differences in electron abstraction abilities of the two oxidants. Thus, the calculations predict that [Fe(IV)=O(Por)Cl](-) is only able to hydroxylate weak C-H bonds, whereas [Fe(IV)=O(Por(+*))Cl] is more versatile.

Discoveries in Bacterial Nucleotide Metabolism

Discoveries in Bacterial Nucleotide Metabolism

Camphor in flavourings and other food ingredients with flavouring properties - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission.

Camphor in flavourings and other food ingredients with flavouring properties - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission.