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.

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.

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: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we used D.

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).

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.

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 (CYP199A4 and CYP199A4), were prepared, characterised and their crystal structures were solved.

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 HaA2, and a range of substituted benzoic acids to investigate different binding modes.

Exploring the substrate specificity of Cytochrome P450.

Cytochromes P450 are enzymes that catalyse the oxidation of a wide variety of compounds that range from small volatile compounds, such as monoterpenes to larger compounds like steroids. These enzymes can be modified to selectively oxidise substrates of interest, thereby making them attractive for applications in the biotechnology industry. In this study, we screened a small library of terpenes and terpenoid compounds against P450 and two P450 mutants, N242A and N242T, that have previously been shown to affect selectivity.

The cubane paradigm in bioactive molecule discovery: further scope, limitations and the cyclooctatetraene complement.

The cubane phenyl ring bioisostere paradigm was further explored in an extensive study covering a wide range of pharmaceutical and agrochemical templates, which included antibiotics (cefaclor, penicillin G) and antihistamine (diphenhydramine), a smooth muscle relaxant (alverine), an anaesthetic (ketamine), an agrochemical instecticide (triflumuron), an antiparasitic (benznidazole) and an anticancer agent (tamibarotene). This investigation highlights the scope and limitations of incorporating cubane into bioactive molecule discovery, both in terms of synthetic compatibility and physical property matching. Cubane maintained bioisosterism in the case of the Chagas disease antiparasitic benznidazole, although it was less active in the case of the anticancer agent (tamibarotenne).

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

P450 and P450 are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein-substrate H bonding has been previously proposed as crucial in the selectivity of P450 oxidations, but not as essential for P450 . To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared.

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants.

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism.

Covering: 2000 up to 2018 The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere.

Direct Observation of an Oxepin from a Bacterial Cytochrome P450-Catalyzed Oxidation.

The cytochromes P450 are hemoproteins that catalyze a range of oxidative C-H functionalization reactions, including aliphatic and aromatic hydroxylation. These transformations are important in a range of biological contexts, including biosynthesis and xenobiotic biodegradation. Much work has been carried out on the mechanism of aliphatic hydroxylation, implicating hydrogen atom abstraction, but aromatic hydroxylation is postulated to proceed differently.

Cytochrome P450cin (CYP176A1).

Cytochrome P450cin (P450cin) (CYP176A1) is a bacterial P450 enzyme that catalyses the enantiospecific hydroxylation of 1,8-cineole to (1R)-6β-hydroxycineole when reconstituted with its natural reduction-oxidation (redox) partner cindoxin, E. coli flavodoxin reductase, and NADPH as a source of electrons. This catalytic system has become a useful tool in the study of P450s as not only can large quantities of P450cin be prepared and rates of oxidation up to 1,500 min(-1) achieved, but it also displays a number of unusual characteristics.

Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation.

Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s.

P450(cin) (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450(cin) does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450(cin) D241N mutant has been produced and found to be analogous to the P450(cam) D251N mutant.

Cytochrome P450 is present in both ferrous and ferric forms in the resting state within intact Escherichia coli and hepatocytes.

Cytochrome P450 enzymes (P450s) are exceptionally versatile monooxygenases, mediating hydroxylations of unactivated C-H bonds, epoxidations, dealkylations, and N- and S-oxidations as well as other less common reactions. In the conventional view of the catalytic cycle, based upon studies of P450s in vitro, substrate binding to the Fe(III) resting state facilitates the first 1-electron reduction of the heme. However, the resting state of P450s in vivo has not been examined.

Oxygen activation by P450(cin): Protein and substrate mutagenesis.

A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450(cin) (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450(cin) N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole.

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme.

Cineole biodegradation: molecular cloning, expression and characterisation of (1R)-6beta-hydroxycineole dehydrogenase from Citrobacter braakii.

The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450(cin) has previously been demonstrated to perform the first oxidation to produce (1R)-6beta-hydroxycineole. In this study, we have cloned cinD from C.

The crystal structure of an LLL-configured depsipeptide substrate analogue bound to isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a non-heme iron(ii) oxidase, which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV) in a remarkable oxidative bicyclisation reaction. The natural substrate for IPNS is the lld-configured tripeptide. lll-ACV is not turned over by the enzyme, but inhibits turnover of the lld-tripeptide.

Structural studies on the reaction of isopenicillin N synthase with a sterically demanding depsipeptide substrate analogue.

Isopenicillin N synthase (IPNS) is a nonheme iron(II)-dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue delta-(L-alpha-aminoadipoyl)-(3S-methyl)-L-cysteine D-alpha-hydroxyisovaleryl ester (A(S)mCOV), designed to intercept the catalytic cycle at an early stage. A(S)mCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a beta-lactam; and the cysteinyl residue is substituted at its beta-carbon, bearing a (3S)-methyl group.

Isopenicillin N synthase mediates thiolate oxidation to sulfenate in a depsipeptide substrate analogue: implications for oxygen binding and a link to nitrile hydratase?

Isopenicillin N synthase (IPNS) is a nonheme iron oxidase that catalyzes the central step in the biosynthesis of beta-lactam antibiotics: oxidative cyclization of the linear tripeptide delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). The ACV analogue delta-L-alpha-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl ester (ACOmC) has been synthesized as a mechanistic probe of IPNS catalysis and crystallized with the enzyme. The crystal structure of the anaerobic IPNS/Fe(II)/ACOmC complex was determined to 1.