Mutant P450 Research Articles

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Combined photoredox/engineered P450 enzymatic direct dioxygen-functionalization of arylalkanes to chiral acyloins.

To date, the examples of difunctionalization of alkanes to directly incorporate two functional groups are very limited. In this study, we combined photoorgano redox catalysis and P450 biocatalysts to obtain dioxygen-functionalization of α/β-C-H bonds of arylalkanes in a straightforward manner. The synthesis of enantiomerically chiral acyloins through a one-pot two-step photoredox/P450-catalyzed cascade reaction is described. Two P450 mutants with stereocomplementary bio-oxidation were obtained using mutagenesis technology and were able to asymmetrically hydroxylate ketones to acyloins with excellent ee values, which were further proved to be efficient on a wide range of substrates. Moreover, a photoredox synthesis of ketones in situ was developed by the direct carbonylation of aromatic methyl C-H bonds and subsequently combined with the aerobic P450-biocatalytic enantioselective hydroxylation of intermediate ketones, thus providing a green and sustainable approach towards optically pure acyloins.

Different Effects of Polymorphic Flavin-Containing Monooxygenase 3 and Cytochrome P450 2A6 Activities on an Index of Arteriosclerosis as a Lifestyle-Related Disease in a General Population in Japan.

The relationships between lifestyle-related diseases and polymorphic drug-metabolizing enzyme activities in the general population in Japan remain unclear. In this study, the relationships between an index of arteriosclerosis and the phenotypic activities of flavin-containing monooxygenase 3 (FMO3) and cytochrome P450 (P450) 2A6 were analysed. Subjects in a general population in Japan (age range 35-97 years, 640 men and 795 women, 12% were current smokers) who took part in a health check program were recruited. Subjects were divided into two groups using the median ankle-brachial pressure index (ABI) score. Subjects harbouring P450 2A6 wild-type allele had a significant age-adjusted odds ratio of 1.3 (95% CI, 1.0-1.6) of having a lower than median ABI score compared with subjects for mutant P450 2A6. For subjects with wild-type FMO3, the odds ratio of 0.89 was not significant. The proportions of P450 2A6 extensive metabolizers varied significantly across the inter-quartile ranges of the ABI scores (p = 0.008). Furthermore, the proportion of subjects with low ABI scores was also dependent on the phenotypic P450 2A6 activity (p = 0.025) as estimated from the P450 2A6 genotype. These results suggest that in a general population in Japan, the ABI score, as a risk index for arteriosclerosis, is associated with the predicted P450 2A6 phenotype but is not associated with FMO3 function. The P450 2A6 wild-type allele may be a possible candidate biomarker for arteriosclerosis in a general population in Japan with a variety of dietary habits.

Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119.

Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon-hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H2O2) as an oxygen and electron donor, but the reaction with H2O2 is inefficient. The development of P450s that can use H2O2 will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H2O2-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher kcat for Amplex Red peroxidation and twofold higher kcat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H2O2. While the Km for H2O2 and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity.

Enzymatic synthesis and chemical inversion provide both enantiomers of bioactive epoxydocosapentaenoic acids

Epoxy PUFAs are endogenous cytochrome P450 (P450) metabolites of dietary PUFAs. Although these metabolites exert numerous biological effects, attempts to study their complex biology have been hampered by difficulty in obtaining the epoxides as pure regioisomers and enantiomers. To remedy this, we synthesized 19,20- and 16,17-epoxydocosapentaenoic acids (EDPs) (the two most abundant EDPs in vivo) by epoxidation of DHA with WT and the mutant (F87V) P450 enzyme BM3 from Bacillus megaterium WT epoxidation yielded a 4:1 mixture of 19,20:16,17-EDP exclusively as (S,R) enantiomers. Epoxidation with the mutant (F87V) yielded a 1.6:1 mixture of 19,20:16,17-EDP; the 19,20-EDP fraction was ∼9:1 (S,R):(R,S), but the 16,17-EDP was exclusively the (S,R) enantiomer. To access the (R,S) enantiomers of these EDPs, we used a short (four-step) chemical inversion sequence, which utilizes 2-(phenylthio)ethanol as the epoxide-opening nucleophile, followed by mesylation of the resulting alcohol, oxidation of the thioether moiety, and base-catalyzed elimination. This short synthesis cleanly converts the (S,R)-epoxide to the (R,S)-epoxide without loss of enantiopurity. This method, also applicable to eicosapentaenoic acid and arachidonic acid, provides a simple, cost-effective procedure for accessing larger amounts of these metabolites.

The cytochrome P450 CYP77A4 is involved in auxin-mediated patterning of the Arabidopsis thaliana embryo.

Metabolism often plays an important role in developmental control, in addition to supporting basal physiological requirements. However, our understanding of this interaction remains limited. Here, we performed quantitative phenome analysis of Arabidopsis thaliana cytochrome P450 mutants to identify a novel interaction between development and metabolism. We found that cyp77a4 mutants exhibit specific defects in cotyledon development, including asymmetric positioning and cup-shaped morphology, which could be rescued by introducing the CYP77A4 gene. Microscopy revealed that the abnormal patterning was detected at least from the 8-cell stage of the cyp77a4 embryos. We next analysed auxin distribution in mutant embryos, as the phenotypes resembled those of auxin-related mutants. We found that the auxin response pattern was severely perturbed in the cyp77a4 embryos owing to an aberrant distribution of the auxin efflux carrier PIN1. CYP77A4 intracellularly localised to the endoplasmic reticulum, which is consistent with the notion that this enzyme acts as an epoxidase of unsaturated fatty acids in the microsomal fraction. We propose that the CYP77A4-dependent metabolic pathway is an essential element for the establishment of polarity in plant embryos.

Functional characterization of cytochrome P450 monooxygenases in the cereal head blight fungus Fusarium graminearum.

Fusarium graminearum is a prominent plant pathogenic fungus causing Fusarium head blight in major cereal crops worldwide. To understand the molecular mechanisms underlying fungal development and virulence, large collections of F. graminearum mutants have been constructed. Cytochrome P450 monooxygenases (P450s) are widely distributed in organisms and are involved in a diverse array of molecular/metabolic processes; however, no systematic functional analysis of P450s has been attempted in filamentous fungi. In this study, we constructed a genome-wide deletion mutant set covering 102 P450s and analyzed these mutants for changes in 38 phenotypic categories, including fungal development, stress responses and responses to several xenobiotics, to build a comprehensive phenotypic dataset. Most P450 mutants showing defective phenotypes were impaired in a single phenotypic trait, demonstrating that our mutant library is a good genetic resource for further fungal genetic studies. In particular, we identified novel P450s specifically involved in virulence (5) and both asexual (1) and sexual development (2). Most P450s seem to play redundant roles in the degradation of xenobiotics in F. graminearum. This study is the first phenome-based functional analysis of P450s, and it provides a valuable genetic resource for further basic and applied biological research in filamentous fungi and other plant pathogens.

Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency.

Cytochrome P450 oxidoreductase (POR) is a 2-flavin protein that transfers electrons from NADPH via its FAD and FMN moieties to all microsomal cytochrome P450 enzymes, including steroidogenic and drug-metabolizing P450s. Defects in the POR gene can cause POR deficiency (PORD), manifested clinically by disordered steroidogenesis, genital anomalies and skeletal malformations. We examined the POR mutant A287P, which is the most frequent cause of PORD in patients of European ancestry and partially disrupts most P450 activities in vitro. Flavin content analysis showed that A287P is deficient in FAD and FMN binding, although the mutation site is distant from the binding sites of both flavins. Externally added flavin partially restored the cytochrome c reductase activity of A287P, suggesting that flavin therapy may be useful for this frequent form of PORD. Transient kinetic dissection of the reaction of POR with NADPH and the reduction in cytochrome c by POR using stopped-flow techniques revealed defects in individual electron transfer steps mediated by A287P. A287P had impaired ability to accept electrons from NADPH, but was capable of a fast FMN → cytochrome c electron donation reaction. Thus the reduced rates of P450 activities with A287P may be due to deficient flavin and impaired electron transfer from NADPH.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450(Prava) bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450(Prava) fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3′-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4′-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000–6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4′ position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

The P450 system and mTORC1 signalling in acne

In this issue, Hellmann-Regen etal. suggested that anti-acne effects of erythromycin and tetracyclines may be related to their inhibitory effect of cytochrome P450-mediated degradation of all-trans-retinoic acid (ATRA). We have recently proposed that all anti-acne agents function by attenuation of increased mTORC1 signalling. This commentary links the P450 system to mTORC1 regulation in acne. Drug-mediated induction of P450 activity or P450 mutants with increased catabolic activity may reduce cellular ATRA levels and FoxO1 expression, thus reducing FoxO-mediated mTORC1 inhibition. In contrast, agents blocking ATRA degradation such as erythromycin and tetracyclines may improve acne by increasing FoxO1 expression with consecutive inhibition of mTORC1 signalling.

Tandem mass spectrometry study of p38α kinase inhibitors and related substances

The p38 mitogen-activated protein kinase α (p38α) is an important drug target widely investigated for therapy of chronic inflammatory diseases. Its inhibitors are rather lipophilic and as such not very favourable lead compounds in drug discovery. Therefore, we explored various approaches to access new chemical space, create diversity, and generate lead libraries with improved solubility and reduced lipophilicity, based on known p38α inhibitors, e.g., BIRB796 and TAK-715. Compound modification strategies include incubation with human liver microsomes and bacterial cytochrome P450 mutants from Bacillus megaterium and treatment by electrochemical oxidation, H2O2, and intense light irradiation. The MS/MS fragmentation pathways of p38α inhibitors and their conversion products have been studied in an ion-trap-time-of-flight MS(n) instrument. Interpretation of accurate mass MS(n) data for four sets of related compounds revealed unexpected and peculiar fragmentation pathways that are discussed in detail. Emphasis is put on the usefulness of HRMS(n)-based structure elucidation in a screening setting and on peculiarities of the fragmentation with regard to the analytes and the MS instrument. In one example, an intramolecular rearrangement reaction accompanied by the loss of a bulky group is observed. For BIRB796, the double-charge precursor ion is used in MS(2), providing a wider range of fragment ions in our instrument. For TAK-715, a number of related compounds could be produced in a large-scale incubation with a Bacillus megaterium mutant, thus enabling comparison of the structure elucidation by (1)H NMR and MS(n). A surprisingly large number of homolytic cleavages are observed. Competition between two fragmentation pathways involving either the loss of CH3(•) or OH(•) radicals was observed for SB203580 and its conversion products.

Electron-Transfer Rates and Dioxygen Reduction Products for Electrode-Wired P450 Mutants

not Available.

Molecular recognition moiety and its target biomolecule interact in switching enzyme activity

We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.

A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid

We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P, were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)2phenA (1) and Ru(phen)2phenA (2) (bpy=bipyridine, phen=1,10-phenanthroline, and phenA=5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding.

Uncovering the Role of Hydrophobic Residues in Cytochrome P450−Cytochrome P450 Reductase Interactions

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Expanding the Substrate Spectrum of Cytochrome P450 2A6 towards Bulky Substrates*

Molecular breeding and biotransformation of cytochrome P450s (P450) are considered two of the emerging areas today. To further expand the substrate spectrum of P450 2A6, N-terminal modification was done to improve its functional expression and consequently, the sensitivity of mutant library screening. Two random libraries screened yielded no gain-of- function mutant, while site-directed mutagenesis generated a new mutant P450 2A6 (N297Q/I300A) with expanded substrate specificity compared with the wild type enzyme and previously reported mutants. The new mutant was able to catalyze the oxidation of 6-benzyloxyindole (6-OBzl-indole) to form colored indigoid dimers as analyzed with mass spectrometry. Both amino acid residue changes were required for the reaction. The results are interpreted in the context of the recently resolved crystal structures of P450 2A6 mutants. The I300A change expands the active-site cavity in both vertical and horizontal directions of the heme plane to be able to fit in substrates of similar nature. The Asn297 is a functionally conserved residue and the N297Q change may be related to hydrogen bond formation.

Did Akhenaten Have the Antley–Bixler Syndrome?

Letters15 December 2009Did Akhenaten Have the Antley–Bixler Syndrome?Irwin M. Braverman, MD and Philip A. Mackowiak, MD, MBAIrwin M. Braverman, MDFrom Yale University School of Medicine, New Haven, CT 06520-8059; and Veterans Affairs Medical Center, Baltimore, MD 21201.Search for more papers by this author and Philip A. Mackowiak, MD, MBAFrom Yale University School of Medicine, New Haven, CT 06520-8059; and Veterans Affairs Medical Center, Baltimore, MD 21201.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-151-12-200912150-00013 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail IN RESPONSE:Our analysis of Akhenaten's kindred revealed 2 abnormalities: craniosynostosis and gynecomastia. These were best explained by 2 genetic disorders: sagittal craniosynostosis and the aromatase syndrome producing excessive estrogen. The Antley–Bixler syndrome (1) was brought to our attention as a possible explanation for our findings. However, the type of cranial deformity in ABS is very different from that found in Akhenaten's kindred, and infertility was not a problem for Akhenaten. However, the gene for the aromatase syndrome regulates a portion of the P450 enzyme system, and the gene for POR deficiency also regulates a portion of the P450 enzyme ...Reference1. Flück CE, Tajima T, Pandey AV, Arlt W, Okuhara K, Verge CF, et al. Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome. Nat Genet. 2004;36:228-30. [PMID: 14758361] CrossrefMedlineGoogle Scholar Author, Article, and Disclosure InformationAffiliations: From Yale University School of Medicine, New Haven, CT 06520-8059; and Veterans Affairs Medical Center, Baltimore, MD 21201.Disclosures: None disclosed. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetailsSee AlsoAkhenaten and the Strange Physiques of Egypt's 18th Dynasty Irwin M. Braverman , Donald B. Redford , and Philip A. Mackowiak Did Akhenaten Have the Antley–Bixler Syndrome? Walter L. Miller Metrics Cited byImpact of type 2 diabetes and microvascular complications on mortality and cardiovascular outcomes in a multiethnic Asian populationIncreased Circulating Visfatin Is Associated With Progression of Kidney Disease in Non-Diabetic Hypertensive Patients 15 December 2009Volume 151, Issue 12Page: 892KeywordsConflicts of interestDNAEnzymesEstrogensGenetic diseasesGenomicsInfertility ePublished: 15 December 2009 Issue Published: 15 December 2009 Copyright & PermissionsCopyright © 2009 by American College of Physicians. All Rights Reserved.PDF downloadLoading ...

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.