Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.     

Biosynthesis of Biscognienyne B Involving a Cytochrome P450-Dependent Alkynylation

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.     

Enzymatic Formation of Indolactam Scaffold by C−N Bond-Forming Cytochrome P450 Oxidases in Teleocidin Biosynthesis

Teleocidins are potent protein kinase C activators, and possess a unique indole-fused nine-membered lactam structure. Teleocidin biosynthesis starts from the formation of a dipeptide by non-ribosomal peptide synthetase (NRPS), followed by oxidative C−N bond formation by a cytochrome P450 oxidase, reverse-prenylation by a prenyltransferase, and methylation-initiated terpene cyclization by a C-methyltransferase. This minireview focuses on recent research progress toward the elucidation of the molecular basis for the remarkable P450-catalyzed intramolecular C−N bond-forming reaction, which is challenging in synthetic chemistry, to generate the indolactam scaffold. In addition, precursor-directed biosynthesis with the promiscuous P450 enzymes led to the formation of a series of unnatural and novel molecular scaffolds, including a sulfur-substituted indolactam with a different conformation from that of indolactam V.     

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis

The 1,3-enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn ( 1 ) is a 1,3-enyne-containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3-enyne biosynthesis in 1 , and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1 . A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis-diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3-enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five-carbon prenyl chain, rather than a polyprenyl chain, to a p-hydroxybenzoic acid acceptor.     

Cytochrome P450cam–monoterpene interactions

The use of the molecular mechanics AMBER force field (FF) to predict product profiles for the hydroxylation of the monoterpenes 1R-camphor, 1S-camphor, 1R-norcamphor, 1S-norcamphor and camphane by the enzyme cytochrome P450cam from the soil bacterium Pseudomonas putida was investigated. Predictions were carried out by applying multiple substrate (starting) orientations in the enzyme pocket in two procedures: a procedure based on molecular dynamics (MD) and a procedure based on short MD simulations followed by geometry optimisations. The latter (GO) procedure is faster and enabled the use of more monoterpene starting orientations. Monoterpene orientations were transformed into product profiles by applying both energetic and geometrical criteria appropriate for the (monoterpene) hydrogen abstraction reaction. Good predictions compared to experimental data were obtained for most compounds in both the MD and GO procedures. Prior to the product profile calculations, the FF was calibrated by reproducing the experimental data for the binding energy of 1R-camphor and 1S-camphor to P450cam and the energy of vaporisation of water. Focus of the calibration was on the value for the scaling factor for the electrostatic interactions.     

How Do Metal Ions Modulate the Rate-Determining Electron-Transfer Step in Cytochrome P450 Reactions?

Cytochrome P450 (CYP450) enzymes play important roles in maintaining human health and their reaction rates are dependent on the first electron transfer from the reduction partner. Interestingly, experimental work has shown that this step is highly influenced by the addition of metal ions. To understand the effect of external perturbations on the CYP450 first reduction step, we have performed a computational study with model complexes in the presence of metal and organic ions, solvent molecules, and an electric field. The results show that these medium-range interactions affect the driving force as well as electron-transfer rates dramatically. Based on the location, distance, and direction of the ions/electric field, the catalytic reaction rates are enhanced or impaired. Calculations on a large crystal structure with bonded alkali metal ions indicated inhibition patterns of the ions. Therefore, we predict that the active forms of the natural CYP450 isozymes will not have more than one alkali metal ion bound in the second-coordination sphere. As such, this study provides an insight into the activity of CYP450 enzymes and the effects of ions and electric field perturbations on their activity.     

Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450 CYP121

Pieces of the puzzle: The first fragment-based approach was used to target cytochrome?P450 enzymes (CYPs) for drug development. The experiments provide new insights into the binding site of the essential Mycobacterium tuberculosis CYP121 enzyme, and resulted in a promising novel lead compound based on fragment merging.     

How Does the Axial Ligand of Cytochrome P450 Biomimetics Influence the Regioselectivity of Aliphatic versus Aromatic Hydroxylation?

How deep is your orbital? Density functional theory studies on the axial ligand effect of aliphatic versus aromatic hydroxylation of ethylbenzene by iron–oxo complexes with a variable axial ligand show that strong (anionic) ligands pull the metal inside the plane of the haeme and destabilise cationic intermediates through orbital interactions (see picture).

     

The Role of Acetylcholine in the Cognitive Function of Frontal Neurons in Monkeys

To study the role of acetylcholine(ACh) in the cognitive function of frontal lobes, ACh and atropine were applied iontophoretically to the task-related neurons recorded from an area around the superior ramus of arcuate sulcus in the frontal cortex of 4 rhesus monkeys during the performance of two tasks. The spontaneous discharges and taskrelated responses of most of the neurons which increased their discharge rate in the delay and differentiation periods of the discrimination response go/no-go task and in the cue and delay periods of the delayed discrimination go/no-go task, were further increased during the application of ACh and suppressed during atropine. These results suggest that ACh may be involved in the excitatory process of the activity of the neurons in the above-mentioned area of frontal cortex to play an important role in the cognitive functions of attention, discrimination and short-term memory.     

Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.     

Effect of Temperature on the Yields of Final Products in the γ-Radiolysis of Formaldehyde Solutions in C1–C3 Alkanols

The effect of temperature on the yields of 1,2-alkanediols in the -radiolysis of the methanol(or ethanol)–formaldehyde (0.9 M) systems and on the yields of 1,2-alkanediols, carbonyl compounds, methanol, and ethanol in the -radiolysis of the 1-propanol(or 2-propanol)–formaldehyde (1 M) systems was studied over the range 373–473 K. It was found that the temperature dependence of the yields of 1,2-butanediol and 2-methyl-1,2-propanediol exhibited maximums at 423 and 373 K, respectively.     

Exploiting a C–N Bond Forming Cytochrome P450 Monooxygenase for C–S Bond Formation

C–S bond formation reactions are widely distributed in the biosynthesis of biologically active molecules, and thus have received much attention over the past decades. Herein, we report intramolecular C–S bond formation by a P450 monooxygenase, TleB, which normally catalyzes a C−N bond formation in teleocidin biosynthesis. Based on the proposed reaction mechanism of TleB, a thiol-substituted substrate analogue was synthesized and tested in the enzyme reaction, which afforded the unprecedented sulfur-containing thio-indolactam V, in addition to an unusual indole-fused 6/5/8-tricyclic product whose structure was determined by the crystalline sponge method. Interestingly, conformational analysis revealed that the SOFA conformation is stable in thio-indolactam V, in sharp contrast to the major TWIST form in indolactam V, resulting in differences in their biological activities.     

The Role of Guest Molecules in the Self-assembly of Metal—ligand Clusters

Abstract

Understanding the self-assembly of nanoscale metal—ligand clusters is an important research area in supramolecular chemistry, especially, if one wishes to develop a truly predictive design strategy for synthesizing these nanoscale clusters. As the building blocks for forming these clusters have become larger and more complex, spacious clusters have been synthesized which often contain large cavities. These assemblies can house guest molecules which play a previously uncharacterized role in the self-assembly processes. We seek to analyze this role: do these guest molecules act as templates? Are the guest molecules necessary for cluster formation? Does the guest drive cluster assemble by forming a stable host—guest complex with the cluster? Must a truly rational design strategy for forming metal—ligand clusters incorporate the use of templates? The role of guest molecules in the self-assembly of nanoscale coordination clusters is reviewed in this article.     

The nature of the P–P bond in carbene-stabilized diphosphorus complex

The nature of the P–P bond in the recently synthesized N-heterocyclic carbene-stabilized diphosphorus complex has been investigated by the built-in fragment-oriented approach using DFT calculations. The result leads us to investigate the stabilization of diphosphorus. It is concluded that a weakening factor, which can be engendered through such methods as orbital mixing, antibonding interaction, or ligand–receptor interactions, is the key to stabilize the highly reactive diphosphorus molecule.     

The Critical Role of Reductive Steps in the Nickel-Catalyzed Hydrogenolysis and Hydrolysis of Aryl Ether C−O Bonds

The hydrogenolysis of the aromatic C−O bond in aryl ethers catalyzed by Ni was studied in decalin and water. Observations of a significant kinetic isotope effect (k_H/k_D=5.7) for the reactions of diphenyl ether under H₂ and D₂ atmosphere and a positive dependence of the rate on H₂ chemical potential in decalin indicate that addition of H to the aromatic ring is involved in the rate-limiting step. All kinetic evidence points to the fact that H addition occurs concerted with C−O bond scission. DFT calculations also suggest a route consistent with these observations involving hydrogen atom addition to the ipso position of the phenyl ring concerted with C−O scission. Hydrogenolysis initiated by H addition in water is more selective (ca. 75 %) than reactions in decalin (ca. 30 %).     

The directive of the protein: how does cytochrome P450 select the mechanism of dopamine formation?

Dopamine can be generated from tyramine via arene hydroxylation catalyzed by a cytochrome P450 enzyme (CYP2D6). Our quantum mechanical/molecular mechanical (QM/MM) results reveal the decisive impact of the protein in selecting the 'best' reaction mechanism. Instead of the traditional Meisenheimer-complex mechanism, the study reveals a mechanism involving an initial hydrogen atom transfer from the phenolic hydroxyl group of the tyramine to the iron-oxo of the compound I (Cpd I), followed by a ring-π radical rebound that eventually leads to dopamine by keto-enol rearrangement. This mechanism is not viable in the gas phase since the O-H bond activation by Cpd I is endothermic and the process does not form a stable intermediate. By contrast, the in-protein reaction has a low barrier and is exothermic. It is shown that the local electric field of the protein environment serves as a template that stabilizes the intermediate of the H-abstraction step and thereby mediates the catalysis of dopamine formation at a lower energy cost. Furthermore, it is shown that external electric fields can either catalyze or inhibit the process depending on their directionality.     

The Role of Sulphonamides and N-Sulphonyl Ketimines/Aldimines as Directing Groups in the Field of C−H Activation

Sulphonamides and N-sulphonyl ketimines/aldimines have turned out to be versatile motifs in the field of synthetic and medicinal chemistry. The field of C−H activation/functionalization flourished remarkably due to their synthetic applicability and directing group plays a remarkable role to achieve regioselectivity in these reactions. The current review summarizes recent tactics by utilizing sulphonamides and N-sulphonyl ketimines/aldimines as directing groups for C−H activation or functionalization. As a directing group, they also facilitate site selectivity and late-stage functionalization of drug molecules in order to construct complex scaffolds of therapeutic importance by C−H activation.     

Free Energy Calculations Give Insight into the Stereoselective Hydroxylation of α-Ionones by Engineered Cytochrome P450 BM3 Mutants

Previously, stereoselective hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N was observed. While both mutants hydroxylate α-ionone in a regioselective manner at the C3 position, M01 A82W catalyzes formation of trans-3-OH-α-ionone products whereas M11 L437N exhibits opposite stereoselectivity, producing trans-(3S,6S)-OH-α-ionone and cis-(3S,6R)-OH-α-ionone. Here, we explore the stereoselective C3 hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N using molecular dynamics-based free energy calculations to study the interaction between the enzyme and both the substrates and the products. The one-step perturbation approach is applied using an optimized reference state for substrates and products. While the free energy differences between the substrates free in solution amount to ～0 kJ mol(-1), the differences in mutant M01 A82W agree with the experimentally obtained dissociation constants K(d). Moreover, a correlation with experimentally observed trends in product formation is found in both mutants. The trans isomers show the most favorable relative binding free energy in the range of all four possible hydroxylated diastereomers for mutant M01 A82W, while the trans product from (6S)-α-ionone and the cis product from (6R)-α-ionone show highest affinity for mutant M11 L437N. Marcus theory is subsequently used to relate the thermodynamic stability to transition state energies and rates of formation.