Oxygen-resistant [FeFe]hydrogenases: new biocatalysis tools for clean energy and cascade reactions.

The use of enzymes to generate hydrogen, instead of using rare metal catalysts, is an exciting area of study in modern biochemistry and biotechnology, as well as biocatalysis driven by sustainable hydrogen. Thus far, the oxygen sensitivity of the fastest hydrogen-producing/exploiting enzymes, [FeFe]hydrogenases, has hindered their practical application, thereby restricting innovations mainly to their [NiFe]-based, albeit slower, counterparts. Recent exploration of the biodiversity of clostridial hydrogen-producing enzymes has yielded the isolation of representatives from a relatively understudied group. These enzymes possess an inherent defense mechanism against oxygen-induced damage. This discovery unveils fresh opportunities for applications such as electrode interfacing, biofuel cells, immobilization, and entrapment for enhanced stability in practical uses. Furthermore, it suggests potential combinations with cascade reactions for CO2 conversion or cofactor regeneration, like NADPH, facilitating product separation in biotechnological processes. This work provides an overview of this new class of biocatalysts, incorporating unpublished protein engineering strategies to further investigate the dynamic mechanism of oxygen protection and to address crucial details remaining elusive such as still unidentified switching hot-spots and their effects. Variants with improved kcat as well as chimeric versions with promising features to attain gain-of-function variants and applications in various biotechnological processes are also presented.

Cascade reactions with two non-physiological partners for NAD(P)H regeneration via renewable hydrogen.

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28 ± 2 nmol NADPH regenerated s-1 mg of hydrogenase-1 (i.e., 1.68 ± 0.12 U mg-1, TOF: 126 ± 9 min-1) and 0.46 ± 0.04 nmol NADH regenerated s-1 mg of hydrogenase-1 (i.e., 0.028 ± 0.002 U mg-1, TOF: 2.1 ± 0.2 min-1), meaning up to 74 mg of NADPH and 1.23 mg of NADH produced per hour by a system involving 1 mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.

A multi-channel microfluidic platform based on human flavin-containing monooxygenase 3 for personalised medicine.

Human flavin-containing monooxygenase 3 (FMO3) is a drug-metabolizing enzyme (DME) which is known to be highly polymorphic. Some of its polymorphic variants are associated with inter-individual differences that contribute to drug response. In order to measure these differences, the implementation of a quick and efficient in vitro assay is highly desirable. To this end, in this work a microfluidic immobilized enzyme reactor (µ-IMER) was developed with four separate serpentines where FMO3 and its two common polymorphic variants (V257M and E158K) were covalently immobilized via glutaraldehyde cross-linking in the presence of a polylysine coating. Computational fluid dynamics simulations were performed to calculate the selected substrate retention time in serpentines with different surface areas at various flow rates. The oxidation of tamoxifen, an anti-breast cancer drug, was used as a model reaction to characterize the new device in terms of available surface area for immobilization, channel coating, and applied flow rate. The highest amount of product was obtained when applying a 10 µL min-1 flow rate on polylysine-coated serpentines with a surface area of 90 mm2 each. Moreover, these conditions were used to test the device as a multi-enzymatic platform by simultaneously assessing the conversion of tamoxifen by FMO3 and its two polymorphic variants immobilized on different serpentines of the same chip. The results obtained demonstrate that the differences observed in the conversion of tamoxifen within the chip are similar to those already published (E158K > WT > V257M). Therefore, this microfluidic platform provides a feasible option for fabricating devices for personalised medicine.

Bioelectrochemical platform with human monooxygenases: FMO1 and CYP3A4 tandem reactions with phorate.

It is highly advantageous to devise an in vitro platform that can predict the complexity of an in vivo system. The first step of this process is the identification of a xenobiotic whose monooxygenation is carried out by two sequential enzymatic reactions. Pesticides are a good model for this type of tandem reactions since in specific cases they are initially metabolised by human flavin-containing monooxygenase 1 (hFMO1), followed by cytochrome P450 (CYP). To assess the feasibility of such an in vitro platform, hFMO1 is immobilised on glassy carbon electrodes modified with graphene oxide (GO) and cationic surfactant didecyldimethylammonium bromide (DDAB). UV-vis, contact angle and AFM measurements support the effective decoration of the GO sheets by DDAB which appear as 3 nm thick structures. hFMO1 activity on the bioelectrode versus three pesticides; fenthion, methiocarb and phorate, lead to the expected sulfoxide products with KM values of 29.5 ± 5.1, 38.4 ± 7.5, 29.6 ± 4.1 µM, respectively. Moreover, phorate is subsequently tested in a tandem system with hFMO1 and CYP3A4 resulting in both phorate sulfoxide as well as phoratoxon sulfoxide. The data demonstrate the feasibility of using bioelectrochemical platforms to mimic the complex metabolic reactions of xenobiotics within the human body.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate.

Out of the five isoforms of human flavin-containing monooxygenase (hFMO), FMO1 and FMO3 are the most relevant to Phase I drug metabolism. They are involved in the oxygenation of xenobiotics including drugs and pesticides using NADPH and FAD as cofactors. Majority of the characterization of these enzymes has involved hFMO3, where intermediates of its catalytic cycle have been described. On the other hand, research efforts have so far failed in capturing the same key intermediate that is responsible for the monooxygenation activity of hFMO1. In this work we demonstrate spectrophotometrically the formation of a highly stable C4a-hydroperoxyflavin intermediate of hFMO1 upon reduction by NADPH and in the presence of O2. The measured half-life of this flavin intermediate revealed it to be stable and not fully re-oxidized even after 30 min at 15 °C in the absence of substrate, the highest stability ever observed for a human FMO. In addition, the uncoupling reactions of hFMO1 show that this enzyme is <1% uncoupled in the presence of substrate, forming small amounts of H2O2 with no observable superoxide as confirmed by EPR spin trapping experiments. This behaviour is different from hFMO3, that is shown to form both H2O2 and superoxide anion radical as a result of ∼50% uncoupling. These data are consistent with the higher stability of the hFMO1 intermediate in comparison to hFMO3. Taken together, these data demonstrate the different behaviours of these two closely related enzymes with consequences for drug metabolism as well as possible toxicity due to reactive oxygen species.

Engineered human CYP2C9 and its main polymorphic variants for bioelectrochemical measurements of catalytic response.

Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min-1) and CYP2C9*2-FLD (12.42 ± 2.17 min-1) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min-1). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy.

N- and S-oxygenation activity of truncated human flavin-containing monooxygenase 3 and its common polymorphic variants.

Human flavin-containing monooxygenase 3 (FMO3) is a membrane-bound, phase I drug metabolizing enzyme. It is highly polymorphic with some of its variants demonstrating differences in rates of turnover of its substrates: xenobiotics including drugs as well as dietary compounds. In order to measure its in vitro activity and compare any differences between the wild type enzyme and its polymorphic variants, we undertook a systematic study using different engineered proteins, heterologously expressed in bacteria, purified and catalytically characterized with 3 different substrates. These included the full-length as well as the more soluble C-terminal truncated versions of the common polymorphic variants (E158K, V257M and E308G) of FMO3 in addition to the full-length and truncated wild-type proteins. In vitro activity assays were performed with benzydamine, tamoxifen and sulindac sulfide, whose products were measured by HPLC. Differences in catalytic properties between the wild-type FMO3 and its common polymorphic variants were similar to those observed with the truncated, more soluble versions of the enzymes. Interestingly, the truncated enzymes were better catalysts than the full-length proteins. The data obtained point to the feasibility of using the more soluble forms of this enzyme for in vitro drug assays as well as future biotechnological applications possibly in high throughput systems such as bioelectrochemical platforms and biosensors.

Biochemical features of dye-decolorizing peroxidases: Current impact on lignin degradation.

Dye-decolorizing peroxidases (DyP) were originally discovered in fungi for their ability to decolorize several different industrial dyes. DyPs catalyze the oxidation of a variety of substrates such as phenolic and nonphenolic aromatic compounds. Catalysis occurs in the active site or on the surface of the enzyme depending on the size of the substrate and on the existence of radical transfer pathways available in the enzyme. DyPs show the typical features of heme-containing enzymes with a Soret peak at 404-408 nm. They bind hydrogen peroxide that leads to the formation of the so-called Compound I, the key intermediate for catalysis. This then decays into Compound II yielding back Fe(III) at its resting state. Each catalytic cycle uses two electrons from suitable electron donors and generates two product molecules. DyPs are classified as a separate class of peroxidases. As all peroxidases they encompass a conserved histidine that acts as the fifth heme ligand, however all primary DyP sequences contain a conserved GxxDG motif and a distal arginine that is their characteristic. Given their ability to attack monomeric and dimeric lignin model compounds as well as polymeric lignocellulose, DyPs are a promising class of biocatalysts for lignin degradation that not only represents a source of valuable fine chemicals, but it also constitutes a fundamental step in biofuels production. Research efforts are envisioned for the improvement of the activity of DyPs against lignin, through directed evolution, ration protein design, or one-pot combination with other enzymes to reach satisfactory conversion levels for industrial applications.

Chimeric cytochrome P450 3A4 used for in vitro prediction of food-drug interactions.

Inhibition of cytochrome P450 (CYP)-mediated drug metabolism by dietary substances is the main cause of drug-food interactions in humans. The present study reports on the in vitro inhibition assays of human CYP3A4 genetically linked to the reductase domain of bacterial BM3 of Bacillus megaterium (BMR) resulting in the chimeric protein CYP3A4-BMR. The activity of this chimeric enzyme was initially measured colorimetrically with erythromycin as the substrate where KM values similar to published data were determined. Subsequently, the inhibition assays with three different dietary products, grapefruit juice, curcumin, and resveratrol, were carried out with the chimeric enzyme both in solution and immobilized on electrode surfaces. For the solution studies, nicotinamide adenine dinucleotide phosphate was added as the electron donor, whereas the need for this cofactor was obviated in the immobilized enzyme as it was supplied by the electrode. Inhibition of the N-demethylation of erythromycin by CYP3A4-BMR chimera was measured at increasing concentrations of the different dietary compounds with calculated IC50 values of 0.5%, 31 µM, and 250 µM for grapefruit juice, curcumin, and resveratrol measured in solution compared with 0.7%, 24 µM, and 208 µM measured electrochemically, respectively. These data demonstrate the feasibility of the use of both CYP3A4-BMR chimera as well as bioelectrochemistry for in vitro studies of not only drug-food interactions but also prediction of adverse drug reactions in this important P450 enzyme.

Production of drug metabolites by human FMO3 in Escherichia coli.

BACKGROUND: In the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites. RESULTS: The effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated. CONCLUSIONS: The study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.

Peroxide-driven catalysis of the heme domain of A. radioresistens cytochrome P450 116B5 for sustainable aromatic rings oxidation and drug metabolites production.

The heme domain of cytochrome P450 116B5 from Acinetobacter radioresistens (P450 116B5hd), a self-sufficient class VII P450, was functionally expressed in Escherichia coli, purified and characterised in active form. Its unusually high reduction potential (-144 ±â€¯42 mV) and stability in the presence of hydrogen peroxide make this enzyme a good candidate for driving catalysis with the so-called peroxide shunt, avoiding the need for a reductase and the expensive cofactor NAD(P)H. The enzyme is able to carry out the peroxide-driven hydroxylation of aromatic compounds such as p-nitrophenol (KM = 128.85 ±â€¯29.51 µM and kcat = 2.65 ±â€¯0.14 min-1), 10-acetyl-3,7-dihydroxyphenoxazine (KM = 6.01 ±â€¯0.32 µM and kcat = 0.33 ±â€¯0.03 min-1), and 3,5,3',5'tetramethylbenzidine (TMB). Moreover, it catalyses different reactions on well-known drugs such as hydroxylation of diclofenac (KM = 49.60 ±â€¯6.30 µM and kcat = 0.06 ±â€¯0.01 min-1) and N-desmethylation of tamoxifen (KM = 57.20 ±â€¯7.90 µM and kcat = 0.79 ±â€¯0.04 min-1). The data demonstrate that P450 116B5hd is an efficient biocatalyst for sustainable applications in bioremediation and human drug metabolite production.

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae.

BACKGROUND: Cytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence. METHODS: Molecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis. RESULTS: A novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids. CONCLUSIONS: The ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce. GENERAL SIGNIFICANCE: The CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.

Uncoupled human flavin-containing monooxygenase 3 releases superoxide radical in addition to hydrogen peroxide.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolizing enzyme capable of performing N- or S-oxidation using the C4a-hydroperoxy intermediate. In this work, we employ both wild type hFMO3 as well as an active site polymorphic variant (N61S) to unravel the uncoupling reactions in the catalytic cycle of this enzyme. We demonstrate that in addition to H2O2 this enzyme also produces superoxide anion radicals as its uncoupling products. The level of uncoupling was found to vary between 50 and 70% (WT) and 90-98% (N61S) for incubations with NADPH and benzydamine over a period of 5 or 20 min, respectively. For the first time, we were able to follow the production of the superoxide radical in hFMO3, which was found to account for 13-18% of the total uncoupling of this human enzyme. Moreover, measurements in the presence or absence of the substrate show that the substrate lowers the level of uncoupling only related to the H2O2 and not the superoxide radical. This is consistent with the entry point of the substrate in this enzyme's catalytic cycle. These findings highlight the importance of the involvement of hFMO3 in the production of radicals in the endoplasmic reticulum, as well as the relevance of single-nucleotide polymorphism leading to deleterious effects of oxidative stress.

A direct time-based ITC approach for substrate turnover measurements demonstrated on human FMO3.

Transient binding events are a challenging issue in enzymology. Here we demostrate a time-based ITC approach to human flavin-containing monooxygenase 3, an important drug metabolising enzyme. We measure kinetic constants and we demonstrate how this approach can be exploited for measuring the inhibiton of the conversion of the key substrate trimethylamine into trimethylamine N-oxide.

Influence of inter-domain dynamics and surrounding environment flexibility on the direct electrochemistry and electrocatalysis of self-sufficient cytochrome P450 3A4-BMR chimeras.

The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megaterium BM3 (BMR). Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. Vmax values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR Vmax values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem.

Binding of methimazole and NADP(H) to human FMO3: In vitro and in silico studies.

Human flavin-containing monooxygenase isoform 3 (hFMO3) is an important hepatic drug-metabolizing enzyme, catalyzing the monooxygenation of nucleophilic heteroatom-containing xenobiotics. Based on the structure of bacterial FMO, it is proposed that a conserved asparagine is involved in both NADP(H) and substrate binding. In order to explore the role of this amino acid in hFMO3, two mutants were constructed. In the case of N61Q, increasing the steric hindrance above the flavin N5-C4a causes poor NADP(H) binding, destabilizing the catalytic FAD intermediate, whereas the introduction of a negatively charged residue, N61D, interferes mainly with catalytic intermediate formation and its stability. To better understand the substrate-enzyme interaction, in vitro as well as in silico experiments were carried out with methimazole as substrate. Methimazole is a high-affinity substrate of hFMO3 and can competitively suppress the metabolism of other compounds. Our results demonstrate that methimazole Pi-stacks above the isoalloxazine ring of FAD in hFMO3, in a similar way to indole binding to the bacterial FMO. However, for hFMO3 indole is found to act as a non-substrate competitive inhibitor. Finally, understanding the binding mode of methimazole and indole could be advantageous for development of hFMO3 inhibitors, currently investigated as a possible treatment strategy for atherosclerosis.

Flavin-Containing Monooxygenase 3 Polymorphic Variants Significantly Affect Clearance of Tamoxifen and Clomiphene.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolising enzyme that oxygenates many drugs and xenobiotics in the liver. This enzyme is also known to exhibit single nucleotide polymorphisms (SNPs) that can alter the rates of monooxygenation of therapeutic agents. The purpose of this study was to investigate the effect of the three common polymorphic variants of hFMO3 (V257M, E158K and E308G) on the metabolism and clearance of three structurally similar compounds: tamoxifen (breast cancer medication), clomiphene (infertility medication) and GSK5182 (antidiabetic lead molecule). For GSK5182, none of the three variants showed any significant differences in its metabolism when compared to the wild-type enzyme. In the case of clomiphene, two of the variants, V257M and E308G, exhibited a significant increase in all the kinetic parameters measured with nearly two times faster clearance. Finally, for tamoxifen, a mixed behaviour was observed; E158K variant showed a significantly higher clearance compared to the wild type, whereas V257M mutation had the opposite effect. Overall, the data obtained demonstrate that there is no direct correlation between the SNPs and the metabolism of these three hFMO3 substrates. The metabolic capacity is both variant-dependent and substrate-dependent and therefore when testing new drugs or administering already approved therapies, these differences should be taken into consideration.

Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops.

Chimerogenesis involving cytochromes P450 is a successful approach to generate catalytically self-sufficient enzymes. However, the connection between the different functional modules should allow a certain degree of flexibility in order to obtain functional and catalytically efficient proteins. We previously applied the molecular Lego approach to develop a chimeric P450 3A4 enzyme linked to the reductase domain of P450 BM3 (BMR). Three constructs were designed with the connecting loop containing no glycine, 3 glycine or 5 glycine residues and showed a different catalytic activity and coupling efficiency. Here we investigate how the linker affects the ability of P450 3A4 to bind substrates and inhibitors. We measure the electron transfer rates and the catalytic properties of the enzyme also in the presence of ketoconazole as inhibitor. The data show that the construct 3A4-5GLY-BMR with the longest loop better retains the binding ability and cooperativity for testosterone, compared to P450 3A4. In both 3A4-3GLY-BMR and 3A4-5GLY-BMR, the substrate induces an increase in the first electron transfer rate and a shorter lag phase related to a domain rearrangements, when compared to the construct without Gly. These data are consistent with docking results and secondary structure predictions showing a propensity to form helical structures in the loop of the 3A4-BMR and 3A4-3GLY-BMR. All three chimeras retain the ability to bind the inhibitor ketoconazole and show an IC50 comparable with those reported for the wild type protein. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Inactivation mechanism of N61S mutant of human FMO3 towards trimethylamine.

Human flavin-containing monooxygenase 3 (hFMO3) catalyses the oxygenation of a wide variety of compounds including drugs as well as dietary compounds. It is the major hepatic enzyme involved in the production of the N-oxide of trimethylamine (TMAO) and clinical studies have uncovered a striking correlation between plasma TMAO concentration and cardiovascular disease. Certain mutations within the hFMO3 gene cause defective trimethylamine (TMA) N-oxygenation leading to trimethylaminuria (TMAU) also known as fish-odour syndrome. In this paper, the inactivation mechanism of a TMAU-causing polymorphic variant, N61S, is investigated. Transient kinetic experiments show that this variant has a > 170-fold lower NADPH binding affinity than the wild type. Thermodynamic and spectroscopic experiments reveal that the poor NADP+ binding affinity accelerates the C4a-hydroperoxyFAD intermediate decay, responsible for an unfavourable oxygen transfer to the substrate. Steady-state kinetic experiments show significantly decreased N61S catalytic activity towards other substrates; methimazole, benzydamine and tamoxifen. The in vitro data are corroborated by in silico data where compared to the wild type enzyme, a hydrogen bond required for the stabilisation of the flavin intermediate is lacking. Taken together, the data presented reveal the molecular basis for the loss of function observed in N61S mutant.

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras.

Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a Vmax increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.