Cloning of cDNA Encoding Choline Monooxygenase from Suaeda liaotungensis and Salt Tolerance of Transgenic Tobacco

Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine be- taine[1]. Two enzymes are involved in the pathway choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spina- cia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42℃), as determined by immunoblot analysis, but did not respond to cold stress (4℃), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.

Monooxygenase Gene from Acinetobacter sp. C42 Inactivates the Acyl Homoserine Lactone Quorum Sensing Signal

[ Objective] This study aimed to investigate the function of aliD gene in the inactivation of AHLs. [ Method ] A bacterial isolate, Acinetobacter sp. CA2 from soil, is capable of inactivation of AHLs. A gene designed as aliD, which is responsible for AHL-quenching activity and exhibits high similarity with Mo- nooxygenase genes, was cloned from the genomic library of Acinetobacter sp. CA2. [ Result ] The aliD gene in-frame deletion mutant, CA2 AliD, impaired its AHLs inactivating function when mixed with N-（3-oxooctanoyl） -L-homosefine lactone （30C8-HSL）. Expression of AliD in plant pathogenic bacterium Pectobacterium ca- rotovorum subsp, carotovorum Z3-3 significantly reduced the AHLs production and the extracellular pectolytic enzyme activities, and attenuated soft rot disease symptoms on the plants tested, including potato, Chinese cabbage, radish and cabbage. [ Conclusion ] Our study suggests that the aliD gene complemented strain CA2-AliD showes a similar AHLs inactivating function.

Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

Transformation and Expression of Choline Monooxygenase(CMO) Gene in Embryogenic Tissue of White Pine(Pinus strobus)

A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was generated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers based on the report sequence of CMO in gene bank. A chimerical gene composed of the cauliflower mosaic virus (CaMV) 35S promoter fused to CMO cDNA and β-glucuronidase (GUS-marker gene) was transferred into Ti-derived disarmed binary vector pBI121. The new vector, p35SCMOp, was transferred into Agrobacterium tumefaciens C58/pMP90 by freeze-thaw method. Somatic embryogenesis (SE) initiation of Pinus. Strobus L. and Pinus.Koraiensis Sieb. et Zucc. depended on the manipulation of plant growth regulator (PGR) concentrations in the GLH culture medium. Transgenic embryos and regenerated plants of two Pine species were produced after co-culture of embryogenic tissue with the disarmed strain of A. tumefaciens C58/pMP90/ p35SCMOp and selected on medium containing 25mg/L kanamycin. The transformed embryogenic tissue was initially confirmed by histochemical GUS assay followed by PCR. One copy of T-DNA was detected by transgenic lines analysis in Pinus. Strobus L. and transgenic plants were regenerated for two species using modified protocols for maturation and germination of somatic embryos.

Effects of tributyltin at environmental levels on monooxygenase system of digestive gland in hard clam Meretrix meretrix

The effects on ethoxyresorufin O-deethylase （EROD）, NADPH-cytochrome c reductase and NADH cytochrorne b5 reductase activities of digestive gland in Meretrix meretrix exposed to tributyltin （TBT） at environmental levels （0.1,1.0,10.0 ng/dm as stannum concentration）,in experimental condition, were evaluated. The EROD, NADH cytochrorne b5 reductase activities were significantly inhibited after exposure to 10.0 ng/dm^3 TBT for 8 and 20 d, the NADPH cytochrorne c reductase activities were significantly inhibited after exposure to 0.1,1.0 and 10.0 ng/dm TBT for 8 d and to 1.0 and 10.0 ng/dm for 20 d, as compared with the matched control, while NADH cytochrorne b5 reductases and NADPH cytochrome c reductase activities were induced after exposure to 10.0 ng/dm^3 TBT for 2 d. The EROD activity in the 10 ng/dm^3 group,and the NADH cytochrome b5 reductases activities in 1.0 and 10.0 ng/dm groups, were significantly induced when transferred to clean recovery tanks for 7 d. The three enzymic activities in the clams exposed to TBT were recovered to the level corresponding to that of the control group after transfer to clean recovery tanks for 20 d. NADPH cytochrome c reductase activity in Meretrix meretrix seems to be more sensitive to exposure of TBT than that of the EROD and NADH cytochrome b5 reductases. The results suggest that induction and inhibition by TBT to the monooxygenase system enzymic activity in Meretrix meretrix would simultaneously exist. The enzymic activities were inhibited by exposure for a long time. The results suggest that inhibition of the monooxygenase system should be an indication of the exposure to environmentally relevant concentrations of TBT for a long time.

A Methane Monooxygenase－like DinuclearIron （Ⅲ） Complex and its Catalytic Ability for Asymmetric Epoxidation of Styrene

A methane monooxygenase-like dinuclear complex of iron (Ⅲ) with a chiral heptadentate ligand has been synthesed and tested for catalytic ability for asymmetric epoxidation of styrene with iodosylbenzene. The results show that the complex exhibits many qualitative features, such as electronic spectrum. catalytic behavior. and asymmetric induction similar to those of methane monooxygenase and catalyzes styrene epoxidation to R- (+)-styrene oxide in 9. 3% yield and 72. 9% optical yield.

Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells

The aim of this study was to examine whether flavin-containing monooxygenase (FMO) protein was expressed in cultured rat brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), and whether N-oxide from the tertiary amine, d-chlorpheniramine, was formed by FMO in rat BMECs. BMECs were isolated and cultured from the brains of three-week-old male Wistar rats. The expression of FMO1, FMO2 and FMO5 proteins was confirmed in rat BMECs by western blotting analysis using polyclonal anti-FMO antibodies, but FMO3 and FMO4 proteins were not found in the rat BBB. Moreover, N-oxide of d-chlorpheniramine was formed in rat BMECs. The intrinsic clearance value for N-oxidation at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent Ki value of 0.53 μmol/L, suggesting that N-oxidation was catalyzed by FMOs in rat BMECs. Although FMO activity in rat BMECs was lower than that in SD rat normal hepatocytes (rtNHeps), we suggest that rat BMECs enzymes can convert substrates of exogenous origin for detoxification, indicating that BMECs are an important barrier for metabolic products besides hepatic cells.

Alkanesulfonate monooxygenase SsuD and its chemicalbiomimetic system

Atkanesulfonate monooxygenase SsuD facilitates the desulfonation reaction of alkane sulfonates to release sulfite and corresponding aldehydes/ketones. Oxygen is activated by the reduced flavin. One oxygen atom is to fi＇om water and the other oxygen atom is to from aldehydes/ketones. The oxidized flavin is regenerated after water is formed. The chemical biomimetic system was established according to the preliminary mechanism of alkanesulfonate monooxygenase and the cyclic mechanism was proposed for the formation ofaldehydes/ketones. Two oxygen atoms from the reduced flavin to form C（4a）-peroxy-flavin. The oxygen atom connected with C（4a） abstracts one electron from the neighbouring oxygen to transfer one oxygen atom to C1 ofalkanesulfonates and abstracts one hydrogen from C1 ofalkanesulfonates to break C 1-H bond. Hydroxy-flavin was produced by the above cyclic mechanism. Alkansulfonate monooxygenase SsuD does not directly involve in the reaction. It only supplies some comfortable environment to facilitate the target reactiorL

Characterization of the P450 Monooxygenase LobP1 as C-32 Hydroxylase in Lobophorin Biosynthesis

Lobophorins(LOBs)belong to a large family of spirotetronate antibiotics with antibacterial and antitumor activities.In this study,we demonstrated the function of LobP1,a P450 monooxygenase encoded in the LOB biosynthetic gene cluster,by in vivo deletion and in vitro biochemical assays.The disruption of lobP1 led to the isolation of three new LOBs derivatives(3-5)and three known ones(6-8)without the hydroxyl group at C-32.LobP1 was shown to have relatively broad substrate scope.Determining the kinetic parameters of LobP1 towards different substrates revealed that LobP1 preferred substrate with a nitrosugar.The new LOBs 3-5 displayed significant antibacterial activities against Bacillus subtilis and Micrococcus luteus with MIC values of 0.125 to 1μg·mL^(-1),and the major product LOB E(6)from the∆lobP1 mutant showed moderate cytotoxic activities against several cancer cell lines.

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase

The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123(1),veramycin A(2),NFAT-133(3)and benwamycin I(4),which were discovered from Streptomyces species and demonstrated with antidiabetic and immunosuppressant activities.Though the biosynthetic pathway of 1-3 was reported as a type I polyketide synthase(PKS),the PKS assembly line was interpreted inconsistently,and it remains a mystery how the compound 3 was generated.Herein,the PKS assembly logic of 1-4 was revised by site-mutagenetic analysis of the PKS dehydratase domains.Based on gene deletion and complementation,the putative P450 monooxygenase nftE1 and metallo-beta-lactamase(MBL)fold hydrolase nftF1 were verified as essential genes for the biosynthesis of 1-4.The absence of nftE1 led to abolishment of 1-4 and accumulation of new products(5-8).Structural elucidation reveals 5-8 as the non-aromatic analogs of 1,suggesting the NftE1-catalyzed aromatic core formation.Deletion of nftF1 resulted in disappearance of 3 and 4 with the compounds 1 and 2 unaffected.As a rare MBL-fold hydrolase from type I PKSs,NftF1 potentially generates the compound 3 through two strategies:catalyze premature chain-offloading as a trans-acting thioesterase or hydrolyze the lactone-bond of compound 1 as an esterase.

The function of steroid terminal monooxygenase in cholesterol or phytosterol degradation by the Mycobacterium neoaurum NRRL B‑3805

Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal medicines.Cytochrome P450s from CYP125 and CYP142 families initiates the metabolism of cholesterol and/or phytosterols.In this study,we demonstrate the functional redundancy and substrate preference of five cytochrome CYP125s and CYP142s by the unmarked cyp genes deletion strains of Mycobacterium neoaurum NRRL B-3805.CYP125-3 was found to be responsible for the catalytic hydroxylation of both cholesterol and the C24-branched side-chains of phytosterols,while CYP142-2 can only oxidize cholesterol and/or cholest-4-en-3-one.The strain harboring the CYP125-3 produced 6.47 g l^(-1)and 5.13 g l^(-1)of 4-androstene-3,17-dione(AD)when 15 g l^(-1)cholesterol or 17.5 g l^(-1)phytosterols used,respectively.While,the strain harboring the CYP142-2 produced 7.10 g l^(-1)AD when 15 g l^(-1)cholesterol used.The other three cytochrome P450s have a smaller contribution for the degradation of cholesterol and phytosterols.CYP125-3 has the hydroxylation activity on more substrates than that of CYP142-2.This study provided possible guidance to improve the efficiency of AD and other metabolites production from different kinds of sterols by the genetic manipulation.

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate(PHB)production of Methylosinus trichosporium OB3b

The coupled effects of nitrogen source and methane monooxygenase（MMO） on the growth and poly-β-hydroxybutyrate（PHB） accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO（s MMO） grew at the highest rate, while N2-fixing bacteria expressing particulate MMO（p MMO） grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content（51.0%） was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB（45.2%） with the expression of p MMO, while N2-fixing bacteria containing p MMO only showed low PHB production capacity（32.1%）. The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of p MMO were greatly improved in the cyclic NO3-N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.

New Mutation of Coenzyme Q10 Monooxygenase 6 Causing Podocyte Injury in a Focal Segmental Glomerulosclerosis Patient

Background:Focal segmental glomerulosclerosis (FSGS)is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function,which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments.The pathogenesis of FSGS has not been completely elucidated;however,recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis.Here,we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. Methods:Using next-generation sequencing and Sanger sequencing,we screened the causative gene that was linked to FSGS in this study.The patient's total blood RNA was extracted to validate the messenger RNA (mRNA)expression of coenzyme Q10 monooxygenase 6(COQ6)and validated it by immunohistochemistry.COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then,F-actin was determined using immunofluorescence staining.Cell apoptosis was evaluated by flow cytometry,the expression of active caspase-3was determined by Western blot,and mitochondrial function was detected by MitoSOX. Results:Using whole-exome sequencing and Sanger sequencing,we screened a new causative gene,COQ6,NM_182480:exonl:c.G41A: p.W14X.The mRNA expression of COQ6 in the proband showed decreased.Moreover,the expression of COQ6,which was validated by immunohistochemistry,also had the same change in the proband.Finally,we focused on the COQ6 gene to clarify the mechanism of podocyte injury.Flow cytometry showed significantly increased in apoptotic podocytes,and Western blotting showed increases in active caspase-3in si-COQ6 podocytes.Meanwhile,reactive oxygen species (ROS)levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. Conclusions:This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6,which could cause respiratory chain defect,increase the generation of ROS,destroy the podocyte cytoskeleton,and induce apoptosis.It provides basic theoretical basis for the screening of FSGS in the future.

A midgut-specific lytic polysaccharide monooxygenase of Locusta migratoria is indispensable for the deconstruction of the peritrophic matrix

Lytic polysaccharide monooxygenases(LPMOs)are important enzymes that boost the hydrolysis of recalcitrant polysaccharides,such as chitin.They are found extensively in different insect species and are classified as auxiliary activities family 15(AA15)LPMOs(LPMO15).Some of them were identified from the insect midgut and proven to act on chitin.However,knowledge about their physiological roles during insect growth and development remains limited.Here,we found that midgut-specific LPMO15s are widely distributed in different insect orders,such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori.Using L.migratoria as a model insect,the function of midgut-specific LmLPMO15-3 during development was investigated.Double-stranded RNA-mediated downregulation of LmLPMO15-3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes.Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix(PM),which suggested that LmLPMO15-3 is essential for the deconstruction of the PM during molting.This study provides direct evidence of the physiological importance of a midgut-specific LPMO15 during insect development.As L.migratoria is one of the most destructive agricultural pests,LmLPMO15-3 is a potential target for pest management.

Cytochrome P450 monooxygenase specific activity reduction in wheat Triticum aestivum induced by soil roxithromycin stress

Roxithromycin, livestock growth promoter, as widely used medicine and arouses concern because its occurrence and persistence in soil environments. However, effects of roxithromycin in higher plants are still vague. Accordingly, we hypothesized that roxithromycin-con- taminated soil may exhibits ecotoxicological effects in wheat （Triticum aestivum）. In this study, effects induced by a gradient concentration of roxithromycin stress （0.01, 0.1, 1, 10, and 100 mg.kg-1） was investigated in a 7-d soil test in T. aestivum. Results indicated that the specific activity of cytochrome P450 （CYP450） monooxygenase was decreased dramatically with the concentration of roxithro- mycin in soil. The IC50 value was 8.78mg.kg-1 of roxithromycin. On the contrary, the growth related endpoints （i.e., the germination percentage, the biomass and the height）, the content related endpoints （i.e., soluble protein content and CYP450 content）, and the superoxide dismutase （SOD） activity failed to reveal the roxithromy- cin-induced effects. Further analysis revealed that the CYP450 monooxygenase specific activity reduction was enzymatic mechanism mediated, other than oxidative stress induced. We conclude that the soil roxithromycin declined the CYP450 monooxygenase activity in T. aestivum by the inhibition of the enzymatic mechanism. Further efforts can include, but are not limited to, investigation of joint effects induced by combined exposure of roxithromycin and the pesticides and evalua- tion of the similar effects in other higher plants.

HMGR, SQS, β-AS, and Cytochrome P450 Monooxygenase Genes in Glycyrrhiza uralensis

Glycyrrhiza uralensis is frequently used in traditional Chinese medicine. This plant contains a large amount of effective constituents, including triterpenoids and flavonoids. Among them, glycyrrhizin is believed to be the marker compound to evaluate the quality of G. uralensis based on Chinese Pharmacopoeia. Many studies showed that glycyrrhizin possesses various pharmacological activities, such as antibacterial, antiviral, antitumor, anti-inflammatory, and immune-stimulating activities. In this paper, we summarized the cloning, characterization, expression, and polymorphism analysis of several functional genes involved in glycyrrhizin biosynthesis in G. uralensis.

Biosynthesis of organic molecules via artificial cascade reactions based on cytochrome P450 monooxygenases

Cytochrome P450 monooxygenases(P450s)play crucial roles in the oxyfunctionalization of non-activated hydrocarbons,thus bridging the gap between simple molecules and high value-added fine chemicals.The introduction of P450s into artificially designed cascade reactions provides an exciting opportunity to accomplish challenging reactions and access organic compounds that cannot be achieved by traditional chemical catalysts or by natural metabolic pathways.The main objective of this review is to provide an overview of different types of artificially designed multi-step cascades in which P450s are involved as key catalysts in the biosynthesis of various organic molecules.The different efforts include in vitro multi-enzymatic biocatalytic cascades,in vivo biocatalytic cascades as well as chemo-enzymatic hybrid cascades.Overall,this work provides an overview of cascade reactions involving P450s with various potential applications for the industrial production of food,cosmetics,polymers and pharmaceuticals.

Chiral model complexes for methane monooxygenase and their asymmetric catalysis of styrene epoxidation

Four chiral μ phenolato dinuclear (VO) 2, Cr 2, Mn 2, and Fe 2 complexes with a phenolic ligand bearing two L phenylanalines as complexing arms have been synthesized and characterized. Asymmetric epoxidations of styrene with these complexes as chiral models of methane monooxygenase (MMO) and iodosylbenzene as an oxygen atom donor have been studied. The results reveal that the Mn 2 complex catalyzes asymmetric epoxidation of styrene with the formation of R (+) styrene oxide in 42.8% e.e. Contrarily, the (VO) 2 analogue complex gives S (-) styrene oxide in 19.2% e.e. EPR investigations suggest that the catalyzed epoxidations with the Mn 2, Cr 2 and Fe 2 complexes proceed via high valent oxometal intermediates; while in the case of the (VO) 2 complex as catalyst, the epoxidation proceeds by a pathway that involves a complex of metal iodine ligand. The catalytic activities of the complexes decrease in a sequence of Mn 2>Cr 2>(VO) 2>Fe 2.

Dioxygen oxidation of hydrocarbons by a methane monooxygenase-like system: diiron complex-O_2-Zn/HOAc-MV^(2+)

The activation of dioxygen and incorporation into hydrocarbons have been achieved under mild conditions by a methane monooxygenase (MMO)-like system using a dinuclear iron complex [Fe2Dhist(OAc)2] BPh4 ·3H2O as the model complex, zinc powder as the electron donor, HOAc as the proton source and methylviologen as the electron transfer agent. The results show that styrene is oxygenated predominantly to styrene oxide (1 396 mol/100 mol of the Fe2 complex), benzaldehyde (16160) and acetophenone (986), and cyclohexane to cyclohexanol (9370) and cyclo-hexanone (2670). EPR studies indicate that the hypervalent ironoxo spiecs FeⅣFeⅣ=O, derived from FeⅢFeⅢ core via reduction, O2-binding and protonation, is the active intermediate which inserts the activated oxygen atom into C=C or C-H bond giving each product. The system closely resembles MMO and its close relative hemerythrin in the aspects of reaction phenomena, EPR characteristics and product distributions. The Mn2 analog cmplex、 Fe-Zn het-erodinuclear complex and mononuclear iron complex show no catalytic activity, indicating that dinuclear iron core is in-despansable to catalytic activity.