Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt.

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

The Oxidation of Oxygen and Sulfur-Containing Heterocycles by Cytochrome P450 Enzymes.

The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a γ-keto-α,ß-unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.

Cymredoxin, a [2Fe-2S] ferredoxin, supports catalytic activity of the p-cymene oxidising P450 enzyme CYP108N12.

Rhodococcus globerulus is a metabolically active organism that has been shown to utilise eucalypt oil as its sole source of carbon and energy. This oil includes 1,8-cineole, p-cymene and limonene. Two identified and characterised cytochromes P450 (P450s) from this organism initiate the biodegradation of the monoterpenes 1,8-cineole (CYP176A1) and p-cymene (CYP108N12). Extensive characterisation has been completed for CYP176A1 and it has been successfully reconstituted with its immediate redox partner, cindoxin, and E. coli flavodoxin reductase. Two putative redox partner genes are encoded in the same operon as CYP108N12 and here the isolation, expression, purification, and characterisation of its specific [2Fe-2S] ferredoxin redox partner, cymredoxin is presented. Reconstitution of CYP108N12 with cymredoxin in place of putidaredoxin, a [2Fe-2S] redox partner of another P450, improves both the rate of electron transfer (from 13 ± 2 to 70 ± 1 µM NADH/min/µM CYP108N12) and the efficiency of NADH utilisation (the so-called coupling efficiency increases from 13% to 90%). Cymredoxin improves the catalytic ability of CYP108N12 in vitro. Aldehyde oxidation products of the previously identified substrates p-cymene (4-isopropylbenzaldehyde) and limonene (perillaldehyde) were observed in addition to major hydroxylation products 4-isopropylbenzyl alcohol and perillyl alcohol respectively. These further oxidation products had not previously been seen with putidaredoxin supported oxidation. Furthermore, when supported by cymredoxin CYP108N12 is able to oxidise a wider range of substrates than previously reported. These include o-xylene, α-terpineol, (-)-carveol and thymol yielding o-tolylmethanol, 7-hydroxyterpineol, (4R)-7-hydroxycarveol and 5-hydroxymethyl-2-isopropylphenol, respectively. Cymredoxin is also capable of supporting CYP108A1 (P450terp) and CYP176A1 activity, allowing them to catalyse the hydroxylation of their native substrates α-terpineol to 7-hydroxyterpineol and 1,8-cineole to 6ß-hydroxycineole respectively. These results indicate that cymredoxin not only improves the catalytic capability of CYP108N12 but can also support the activity of other P450s and prove useful for their characterisation.

Synthesis of substituted norcaranes for use as probes of enzyme mechanisms.

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.

Differentiating Dyes: A Spectroscopic Investigation into the Composition of Scarlet Bloodroot (Haemodorum coccineum R.Br.) Rhizome.

Haemodorum coccineum, commonly known as scarlet bloodroot, is a plant native to New Guinea and the northern most parts of Australia. The highly coloured H. coccineum is used by communities in Larrakia country for dyeing garments and occasionally to treat snake bites. Previous studies into H. coccineum have focused on its taxonomic classification, with this being the first evaluation of the chemical composition of the plant. Haemodoraceae plants are reported to contain phenylphenalenones (PhPs), which are highly conjugated polycyclic oxygenated aromatic hydrocarbons. We report the characterisation of 20 compounds extracted from the rhizome of H. coccineum: four sugars and 16 compounds belonging to the PhP family. The compounds include five aglycones and seven glycosylated compounds, of which four contain malonate esters in their structures. Characterisation of these compounds was achieved through 1D and 2D NMR, MS analysis and comparison to the known phytochemistry of other species from the Haemodorum genus. Preliminary anti-microbial activity of the crude extract shows significant inhibition of the growth of both gram-positive and gram-negative bacteria, but no activity against Candida albicans.

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions.

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)-H2 O2 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)-H2 O2 complex which would be more abundant in the peroxide-driven reactions.

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering.

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.

CYP108N12 initiates p-cymene biodegradation in Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) isolated from soil beneath Eucalyptus sp. was found to live on the monoterpenes 1,8-cineole, p-cymene and (R)- and (S)-limonene as sole sources of carbon and energy. Previous metabolic studies revealed that R. globerulus is capable of living on 1,8-cineole, the main monoterpene component of eucalyptus essential oil through the activity of cytochrome P450cin (CYP176A1) [1]. Genomic sequencing of R. globerulus revealed a novel putative cytochrome P450 (CYP108N12) that shares 48% sequence identity with CYP108A1 (P450terp) from Pseudomonas sp., an α-terpineol hydroxylase. Given the sequence similarity between CYP108N12 and P450terp, it was hypothesised that CYP108N12 may be responsible for initiating the biodegradation of a monoterpene structurally similar to α-terpineol such as (R)-limonene, (S)-limonene or p-cymene. Encoded within the operon containing CYP108N12 were two putative bacterial P450 redox partners and putative alcohol and aldehyde dehydrogenases, suggesting a complete catalytic system for activating these monoterpenes. Binding studies revealed that p-cymene and (R)- and (S)-limonene all bound tightly to CYP108N12 but α-terpineol did not. A catalytically active system was reconstituted using the non-native redox partner putidaredoxin and putidaredoxin reductase that act with CYP101A1 (P450cam) from Pseudomonas. This reconstituted system catalysed the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol, and (R)- and (S)-limonene to (R)- and (S)-perillyl alcohol, respectively. R. globerulus was successfully grown on solely p-cymene, (R)-limonene or (S)-limonene. CYP108N12 was detected when R. globerulus was grown on p-cymene, but not either limonene enantiomer. The native function of CYP108N12 is therefore proposed to be initiation of p-cymene biodegradation by methyl oxidation and is a potentially attractive biocatalyst capable of specific benzylic and allylic hydroxylation.

To Be, or Not to Be, an Inhibitor: A Comparison of Azole Interactions with and Oxidation by a Cytochrome P450 Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe-N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe-N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.

Synthesis of obtusifoliol and analogues as CYP51 substrates.

Sterol 14α-demethylases (CYP51s) are a ubiquitous superfamily of cytochrome P450 enzymes that play an essential role in sterol biosynthesis. As fungal CYP51s are the target of azole-based antifungal agents, which are facing the problem of increasing resistance, the substrate specificity of this enzyme subclass has recently garnered significant attention. Herein we report the first chemical synthesis of the final endogenous substrate of this enzyme class, obtusifoliol, in 1.3% yield across ten steps from a commercially available lanosterol mixture. Intermediates along this pathway provide a basis for further derivatisation of the sterol skeleton and future investigation into CYP51 inhibition to overcome pathogens' azole resistance.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases.

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-, ß-, 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.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes.

Oxygen surrogates (OSs) have been used to support cytochrome P450 (P450) enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application, little is known of the preference of individual P450s for different OSs or the substrate dependence of OS-supported activity. Furthermore, the biocatalytic potential of OSs other than cumene hydroperoxide (CuOOH) and hydrogen peroxide (H2O2) is yet to be explored. Here, we investigated the ability of the major human drug-metabolizing P450s, namely CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2, to use the following OSs: H2O2, tert-butyl hydroperoxide (tert-BuOOH), CuOOH, (diacetoxyiodo)benzene, and bis(trifluoroacetoxy)iodobenzene. Overall, CuOOH and tert-BuOOH were found to be the most effective at supporting these P450s. However, the ability of P450s to be supported by OSs effectively was also found to be highly dependent on the substrate used. This suggests that the choice of OS should be tailored to both the P450 and the substrate under investigation, underscoring the need to employ screening methods that reflect the activity toward the substrate of interest to the end application. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450) enzymes can be supported by different oxygen surrogates (OSs), avoiding the need for a redox partner and costly NADPH. However, few data exist comparing relative activity with different OSs and substrates. This study shows that the choice of OS used to support the major drug-metabolizing P450s influences their relative activity and regioselectivity in a substrate-specific fashion and provides a model for the more efficient use of P450s for metabolite biosynthesis.

Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes.

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Graphic abstract.

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues.

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown. We show that combining peptide synthesis with enzymatic cyclisation enables the formation of novel examples of GPAs and provides an indication of the utility of these crucial enzymes. By accessing the biosynthetic process in vitro, we identified peptide modifications that are enzymatically tolerated and can also reveal the mechanistic basis for substrate intolerance where present. Using this approach, we next specifically activated modified residues within GPAs for functionalisation at previously inaccessible positions, thereby offering the possibility of late-stage chemical functionalisation after GPA cyclisation is complete.

Rearrangement-Free Hydroxylation of Methylcubanes by a Cytochrome P450: The Case for Dynamical Coupling of C-H Abstraction and Rebound.

The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported k = 2.9 × 1010 s-1 at 25 °C) through scission of two bonds of the cube. The rearrangement has previously been used as a mechanistic probe to detect radical-based pathways in enzyme-catalyzed C-H oxidations. This paper reports the discovery of highly selective cytochrome P450-catalyzed methylcubane oxidations which notionally proceed via cubylmethyl radical intermediates yet are remarkably free of rearrangement. The bacterial cytochrome P450 CYP101B1 from Novosphingobium aromaticivorans DSM 12444 is found to hydroxylate the methyl group of a range of methylcubane substrates containing a regio-directing carbonyl functionality at C-4. Unlike other reported P450-catalyzed methylcubane oxidations, the designed methylcubanes are hydroxylated with high efficiency and selectivity, giving cubylmethanols in yields of up to 93%. The lack of cubane core ring-opening implies that the cubylmethyl radicals formed during these CYP101B1-catalyzed hydroxylations must have very short lifetimes, of just a few picoseconds, which are too short for them to manifest the side reactivity characteristic of a fully equilibrated P450 intermediate. We propose that the apparent ultrafast radical rebound can be explained by a mechanism in which C-H abstraction and C-O bond formation are merged into a dynamically coupled process, effectively bypassing a discrete radical intermediate. Related dynamical phenomena can be proposed to predict how P450s may achieve various other modes of reactivity by controlling the formation and fate of radical intermediates. In principle, dynamical ideas and two-state reactivity are each individually able to explain apparent ultrashort radical lifetimes in P450 catalysis, but they are best considered together.

Significance of Protein-Substrate Hydrogen Bonding for the Selectivity of P450-Catalysed Oxidations.

P450cin and P450cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein-substrate H bonding has been previously proposed as crucial in the selectivity of P450cin oxidations, but not as essential for P450cam . To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein-substrate H bond to selectivity in both P450cin and P450cam . The H bonding in P450cam was re-examined using camphane, another substrate for which the protein-substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein-substrate H bond was not a factor in substrate affinity for the enzyme.