Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula.

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.

SFRP2/DPP4 and FMO1/LSP1 Define Major Fibroblast Populations in Human Skin.

Fibroblasts produce matrix, regulate inflammation, mediate reparative processes, and serve as pluripotent mesenchymal cells. Analyzing digested normal human skin by single-cell RNA sequencing, we explored different fibroblast populations. T-distributed stochastic neighbor embedding and clustering of single-cell RNA sequencing data from six biopsy samples showed two major fibroblast populations, defined by distinct genes, including SFRP2 and FMO1, expressed exclusively by these two major fibroblast populations. Further subpopulations were defined within each of the SFRP2 and FMO1 populations and five minor fibroblast populations, each expressing discrete genes: CRABP1, COL11A1, FMO2, PRG4, or C2ORF40. Immunofluorescent staining confirmed that SFRP2 and FMO1 define cell types of dramatically different morphology. SFRP2+ fibroblasts were small, elongated, and distributed between collagen bundles. FMO1+ fibroblasts were larger and distributed in both interstitial and perivascular locations. Differential gene expression by SFRP2+, FMO1+, and COL11A1+ fibroblasts suggests roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation, respectively.

Cloning of Toluene 4-Monooxygenase Genes and Application of Two-Phase System to the Production of the Anticancer Agent, Indirubin.

Indirubin is a strong inhibitor of several eukaryotic cell signaling pathways and shows promise as a treatment for myelocytic leukemia and Alzheimer's disease. The tmoABCDEF operon, encoding the components of a novel toluene 4-monooxygenase from the paint factory soil isolate, Pseudomonas sp. M4, was cloned and expressed in Escherichia coli. E. coli::pKSR12 expressing the tmo genes was used to develop a two-phase [dioctyl phthalate (DOP)/aqueous medium] culture system that was optimized to obtain maximal yields of indirubin from the starting substrate, indole. DOP was used as the organic phase to solubilize and sequester the toxic indole substrate, making possible the use of high indole concentrations that would otherwise interfere with growth in aqueous media. A 50 % (v/v) DOP two-phase system using tryptophan medium containing 3 mM cysteine, 5 mM indole, and 1 mM isatin yielded 102.4 mg/L of indirubin with no conversion of indole to the less valuable alternate product, indigo.

Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability.

The consequences of ketocarotenoid production in transgenic tobacco (Nicotiana tabacum) plants expressing a Chlamydomonas reinhardtii gene encoding a ß-carotene ketolase were examined concerning the functionality of the photosynthetic apparatus. T1 plants produced less photosynthetic pigments per dry weight, but Chl a/Chl b ratios remained unchanged. Almost as much ketocarotenoids as accessory xanthophylls accumulated per Chl a molecule. These ketocarotenoids were found mainly in the thylakoid membranes, but were not functionally bound to light-harvesting complexes, although LHCII is known to be able to bind astaxanthin. On the contrary, high amounts of ketocarotenoids probably changed the properties of the lipid phase of the thylakoids, thereby reducing the stability of photosystem II supercomplexes and LHCII trimers and ultimately decreasing grana formation. In addition, photosystem II function in electron transport was impaired, and plants exhibited less non-photochemical quenching compared to wild-type plants. Thus, in order not to disturb vital functions of the plants, production of astaxanthin and other nutritionally valuable ketocarotenoids apparently requires ways to sequester the additional carotenoids to plastoglobuli.

Competition between metals for binding to methanobactin enables expression of soluble methane monooxygenase in the presence of copper.

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

Temporomandibular Disorders in a Sample Population of the Brazilian Northeast

Temporomandibular disorder (TMD) is a common condition. This study is part of a research group and it investigated the prevalence of TMD and myofascial pain and its association with gender, age and socioeconomic class. The sample comprised 100 subjects, aged 15 to 70, users of the Family Health Units' services, in the city of Recife, PE, Brazil. The TMD degree was evaluated using the Research Diagnostic Criteria for TMD and socioeconomic class by the Economic Classification Criteria Brazil. Categorical variables were analyzed by chi-square test for proportions and Fisher's exact test for 2x2 tables, and binary logistic analysis to track the relationship between the independent and dependent variables. According to the results, 42% of the subjects had TMD and 14% myofascial pain. No statistically significant association could be found between TMD and gender or socioeconomic class, but it was found to have statistically significant association with age, and myofascial pain was associated with socioeconomic class. Considering that the results of the present study should be confirmed by further studies and the fact that this was a pilot study, the prevalence must be analyzed with caution.

Disfunção temporomandibular (DTM) é uma condição comum. Este estudo é parte de um grupo de pesquisa e investigou a prevalência de DTM e dor miofascial e suas associações com sexo, idade e classe socioeconômica. A amostra foi composta por 100 indivíduos, com idades entre 15 e 70 anos, usuários das Unidades de Saúde da Família, na cidade de Recife, PE. O grau de DTM foi avaliado usando os Critérios de Diagnósticos Científicos em DTM, e classe socioeconômica com o Critério de Classificação Econômica Brasil. As variáveis categóricas foram analisadas pelo teste do qui-quadrado para proporções e teste exato de Fisher para tabelas 2x2, e a análise logística binária para traçar a relação entre as variáveis independentes e dependentes. De acordo com os resultados, 42% dos indivíduos tinham DTM e 14% dor miofascial. Não houve associação estatisticamente significativa entre DTM e sexo ou classe socioeconômica, mas houve associação estatisticamente significativa com a idade e a dor miofascial foi associada com a classe socioeconômica. Considerando-se que os resultados do presente estudo devam ser confirmados em outros estudos e por causa de sua natureza piloto, a prevalência deve ser analisada com cautela.

Methanobactin and MmoD work in concert to act as the 'copper-switch' in methanotrophs.

Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.

Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

AtMYB2 transcription factor can interact with the CMO promoter and regulate its downstream gene expression.

The pC5 promoter, a region of the choline monooxygenase (CMO) promoter, contains an AtMYB2 transcription factor recognition sequence, TAACCA, and we have examined the interaction between AtMYB2 and the pC5 promoter. The AtMYB2 gene was cloned from Arabidopsis, expressed in Escherichia coli and transferred into pC5-GUS transgenic tobacco plants. Using an electrophoretic mobility shift assay, the AtMYB2 fusion protein binds to the TAACCA sequence in the pC5 promoter. As GUS activity was higher in pC5-GUS/AtMYB2 transgenic tobacco than in pC5-GUS plants, the AtMYB2 protein can bind to the CMO promoter in vitro and activate the transcription of the GUS reporter gene in vivo. The AtMYB2 transcription factor can therefore interact with the CMO promoter directly and regulate its downstream gene expression.

Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver.

Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

Tissue-specific expression of the PNZIP promoter is mediated by combinatorial interaction of different cis-elements and a novel transcriptional factor.

Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.

Trisindoline synthesis and anticancer activity.

Expression of a Rhodococcus-derived oxygenase gene in Escherichia coli yielded indigo metabolites with cytotoxic activity against cancer cells. Bioactivity-guided fractionation of these indigo metabolites led to the isolation of trisindoline as the agent responsible for the observed in vitro cytotoxic activity against cancer cells. While the cytotoxicity of etoposide, a common anticancer drug, was dramatically decreased in multidrug-resistant (MDR) cancer cells compared with treatment of parental cells, trisindoline was found to have similar cytotoxicity effects on both parental and MDR cell lines. In addition, the cytotoxic effects of trisindoline were resistant to P-glycoprotein overexpression, one of the most common mechanisms of drug resistance in cancer cells, supporting its use to kill MDR cancer cells.

Nicotiana glauca engineered for the production of ketocarotenoids in flowers and leaves by expressing the cyanobacterial crtO ketolase gene.

Nicotiana glauca is a tobacco species that forms flowers with carotenoid-pigmented petals, sepals, pistil, ovary and nectary tissue. The carotenoids produced are lutein, ss-carotene as well as some violaxanthin and antheraxanthin. This tobacco species was genetically modified for ketocarotenoid biosynthesis by transformation with a cyanobacterial crtO ketolase gene under the 35S CaMV promoter. In the transformants, ketocarotenoids were detected in both leaves and flowers. Although astaxanthin was not detected other ketocarotenoids such as 4'-ketolutein, echinenone, 3'-hydroxyechinenone and 4-ketozeaxanthin were present. Accumulation of ketocarotenoids in leaves decreased their photosynthetic efficiency moderately. Under the green house conditions used no impairment of growth and development compared to the wild type was observed. In the crtO-transformants, an unexpected up-regulation of total carotenoid biosynthesis in leaves and especially in flower petals was observed. This led to a total ketocarotenoid concentration in leaves of 136.6 (young) or 156.1 (older) mug/g dry weight and in petals of 165 mug/g dry weight. In our engineered plants, the ketocarotenoid pathway is one step short of astaxanthin. Strategies are discussed to improve N. glauca flowers as a biological system for astaxanthin.

Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris.

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.

A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum.

The Aspergillus nidulans velvet (veA) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the veA homologue AcveA from Acremonium chrysogenum, the major producer of the beta-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate that the nucleus-localized AcVEA polypeptide controls the transcriptional expression of six cephalosporin C biosynthesis genes. The most drastic reduction in expression is seen for cefEF, encoding the deacetoxycephalosporine/deacetylcephalosporine synthetase. After 120 h of growth, the cefEF transcript level is below 15% in both disruption strains compared to the wild type. These transcriptional expression data are consistent with results from a comparative and time-dependent high-performance liquid chromatography analysis of cephalosporin C production. Compared to the recipient, both disruption strains have a cephalosporin C titer that is reduced by 80%. In addition to its role in cephalosporin C biosynthesis, AcveA is involved in the developmentally dependent hyphal fragmentation. In both disruption strains, hyphal fragmentation is already observed after 48 h of growth, whereas in the recipient strain, arthrospores are not even detected before 96 h of growth. Finally, the two mutant strains show hyperbranching of hyphal tips on osmotically nonstabilized media. Our findings will be significant for biotechnical processes that require a defined stage of cellular differentiation for optimal production of secondary metabolites.

Effects of diabetes on rabbit kidney and lung CYP2E1 and CYP2B4 expression and drug metabolism and potentiation of carcinogenic activity of N-nitrosodimethylamine in kidney and lung.

There are limited number of studies regarding the influence of diabetes on the regulation of cytochrome P450s and associated drug metabolizing enzyme activities especially in extrahepatic tissues such as kidney. However, there is almost no such study in lung. Alloxan-induced diabetes did not change CYP2B4 expression as measured with immunoblot analysis and associated enzyme, benzphetamine N-demethylase, activity in rabbit kidney and lung. Induction of cytochrome P4502E1 by diabetes was identified by immunochemical detection on Western blots in the lung and kidney microsomes of rabbits. In parallel to CYP2E1 induction, aniline 4-hydroxylase and p-nitrophenol hydroxylase activities were markedly increased in diabetic rabbit lung and kidney. CYP2B4 and CYP2E1 dependent drug metabolism did not show any tissue variation in diabetic rabbit. These findings are in contrast to those of rats, mice and hamster. The results of the present work, in combination with those of the previous work [Arinç, E., Arslan, S., Adali, O., 2005. Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver. Arch. Toxicol. 79, 427-433], indicate the existence of species-dependent response of CYP-dependent drug metabolizing enzymes to diabetes. A procarcinogen and food contaminant, N-nitrosodimethylamine (NDMA), is converted to its carcinogenic form after it is activated with NDMA N-demethylase. In the current study, a statistically significant increase of liver, kidney and lung NDMA N-demethylase activity associated with CYP2E1 was shown in diabetic rabbit. Thus, it is expected that, the risk of nitrosamine induced carcinogenesis will be greater in liver, kidney and lung of the diabetic subjects.

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase.

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BK(Ca)) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK(Ca) channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10(-5) M) increased 14,15-EET release twofold and BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK(Ca) channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta).

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a beta-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting beta-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4'-beta-ionone ring oxygenase.

Characterization of kinetics and products of the Baeyer-Villiger oxygenase MtmOIV, the key enzyme of the biosynthetic pathway toward the natural product anticancer drug mithramycin from Streptomyces argillaceus.

MtmOIV, the key oxygenase of the mithramycin biosynthetic pathway in Streptomyces argillaceus, was proven to act initially as Baeyer-Villiger monooxygenase, but may also catalyze various follow-up reaction steps. The reaction of the overexpressed pure His6-tagged enzyme with its substrate premithramycin B was studied. Various intermediates and products were isolated and physicochemically characterized, several of them being previously unknown compounds. This is the first example in which a bacterial enzyme was unequivocally proven to act as Baeyer-Villigerase with its natural substrate, that is, in its natural context.