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PubMed1993

YM934, a novel K+ channel opener, activates ATP-sensitive K+ channels in cardiac myocytes.

2-(3,4-Dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl)pyridin e N-oxide (YM934) is a newly synthesized benzoxazin. The effects of YM934 on ATP-sensitive K+ (KATP) channels in guinea pig cardiac ventricular myocytes and in an insulin-secreting cell line, HIT T15 beta-cells, were examined using the gigaohm-seal patch-clamp techniques. Under the whole-cell clamp condition, YM934 induced in ventricular myocytes a time-independent, glibenclamide-sensitive K+ current in a concentration-dependent fashion (EC50 = approximately 3 microM). On formation of inside-out patches in ATP-free solution, the KATP channel current abruptly appeared and then ran down. YM934 was applied to inside-out patches before, during and after channel "run-down." Because nucleoside diphosphates, such as uridine diphosphate (UDP), can induce channel openings after complete run-down, the effects of YM934 on the UDP-induced channel openings were also examined. Before run-down, YM934 enhanced KATP channel activity by decreasing the sensitivity of channels to intracellular ATP. YM934 also enhanced the partially run-down channel, even in the absence of ATP. After run-down, YM934 had no effect but could enhance the UDP-induced KATP channel openings. These effects of YM934 on cardiac KATP channels were similar to those of pinacidil and lemakalim. In HIT T15 beta-cells, 100 microM YM934 was ineffective in both cell-attached and inside-out patch configurations, suggesting the tissue-specific nature of the action of this novel K+ channel opener.

Adenosine Triphosphate↗
PubMed1997

Synthesis, stability and anticonvulsant activity of two new GABA prodrugs.

4-(3,4-Dihydro-2,4-dioxo-2H-1,3-benzoxazin-3-yl)-butyric acid (7) and its ester 6, two potential gamma-aminobutyric acid (GABA) prodrugs, were synthesized and studied to determine their stability in aqueous buffer and their susceptibility to undergo enzymatic hydrolysis in vitro (mouse plasma). Both compounds were fairly stable in aqueous media, (t1/2 = 68.2 h and 25.7 h, respectively). The 3,4-dihydro-2,4-dioxo-2H-1,3-benzoxazine ring underwent enzymatic hydrolysis (t1/2 = 5.8 h) in compound 7, whereas in compound 6 it seemed not to be opened by mouse plasma esterases within the observation time (3h). Both compounds were tested for their anticonvulsant activity in pentetrazole (PTZ) treated mice, and showed significant activity. Compound 7, administered as sodium salt 8, was active at relatively low doses and can be considered a very interesting GABA prodrug.

Animals↗
PubMed1999

The hydroxamic acid pathway.

An important component of general defence mechanisms of plants are toxic secondary metabolites that function as natural pesticides. The cyclic hydroxamic acids DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) play an important role in the chemical defence of cereals against pests such as insects and pathogenic fungi and bacteria. Five genes that are clustered on chromosome four are sufficient to encode the enzymes to synthesize DIBOA. The first gene in the pathway, Bx1, encodes an enzyme resembling a tryptophan synthase alpha subunit that catalyses the formation of indole and thereby establishes the branchpoint that leads to the secondary metabolites. Four cytochrome P450-dependent monooxygenases encoded by Bx2-Bx5 catalyse consecutive hydroxylations to form DIBOA. This pathway can be generalized for grasses, since identical enzyme activities have been found in rye. The pathway is relatively short and begins with a metabolite ubiquitous to plants. Therefore DIBOA biosynthesis could be introduced into other plant species to confer improved disease resistance.

Hydroxamic Acids↗
PubMed1993

Synthesis and molluscicidal activity of new derivatives of 1-(hydroxy/substituted phenyl)-3-arylpropenones.

Several 1-(hydroxy/substituted phenyl)propenones were tested as molluscicidal agents among which the 1-(2-hydroxy/substituted phenyl)-3-(2-furyl)propenones 1a-c show the most promising results. In an attempt to improve their activity, new dihydropyrazolo[1,5-c][1,3]benzoxazines, their thio and their dehydrogenated derivatives were prepared. The pyrazolo[1,5-c][1,3]benzoxazines 4a,b were hydrolyzed affording the 3-(2-furyl)-5-(2-hydroxy/substituted phenyl) pyrazoles 8a,b. The hydroxyimino derivatives 10a-c were synthesized together with their corresponding isoxazole derivatives 11a-c. -Molluscicidal assay indicated that the oximes 10b,c, the isoxazole 11b, the pyrazole 8b, and its N-carbamoyl derivative 9b are most effective. They have in common the conjugated system shown in Fig. 1 which is presumably the active core.

Animals↗
PubMed2000

3,4-Dihydro-3,4,6-trimethyl-2H,8H-pyrano-

The synthesis of a novel "reporter group" reagent-3,4-dihydro-3,4,6-trimethyl-2H,8H-pyrano-[3,2g]-1,3-benzoxazin-2,8-dione (DTPBD)-is described. This compound has a cyclic carbamate functionality and thus, like the previously used 3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (DMNB), has the potential to label an esterase such as chymotrypsin. In so doing, DTPBD would incorporate into the protein a covalently linked 4-methylumbelliferone derivative, thus providing a sensitive reporter group that is both chromophoric and fluorescent. Experiments show that DTPBD reacts with chymotrypsin in the predicted manner, except that the labeling process is freely reversible (unlike the case with DMNB). 4-Methylumbelliferyl acetate (which is structurally closely related to DTPBD) is a good substrate for chymotrypsin. The rate of acylation of the enzyme is about an order of magnitude faster than with p-nitrophenyl acetate (which in turn is structurally related to DMNB), an unexpected observation in view of the relative leaving-group abilities of the groups concerned. The results suggest that these various modifiers and substrates show subtle but significant differences in the way they position themselves in chymotrypsin's binding site. Copyright 2000 Academic Press.

Journal Article↗
PubMed2005

Synthesis and evaluation of new quinazolone derivatives of nalidixic acid as potential antibacterial and antifungal agents.

In continuation of our work on synthesis of biheterocycles carrying the biodynamic heterocyclic systems at position 3, a series of new nalidixic acid derivatives having quinazolones moiety were synthesised to achieve enhanced biological activity and wide spectrum of activity. Nalidixic acid was first converted into its acid chloride using thionyl chloride as an acylating agent at laboratory temperature. Later it was converted to methyl ester. Nalidixoyl chloride formed vigorously reacts with methanol to give a methyl ester of nalidixic acid. The ester on addition of hydrazine hydrate furnished nalidixic acid hydrazide. Appropriate anthranilic acid was refluxed with acetic anhydride to form Benzoxazine/Acetanthranil. 5-iodo-derivative of anthranilic acid was prepared and also utilised to obtain 6-iodo-Benzoxazine/Acetanthranil. Also, 6-nitro-Benzoxazine/Acetanthranil was obtained by nitration of acetanthranil using conc. H(2)SO(4) and fuming HNO(3). Equimolar proportions of the appropriate synthesised acetanthranils and nalidixic acid hydrazide in the presence of ethanol were refluxed to synthesise quinazolones. Elemental analysis and IR spectra confirmed nalidixic acid hydrazide formation. The structures of the compounds obtained have been established on the basis of Spectral (IR, (1)H NMR and mass) data. The current study also involves in vitro antimicrobial screening (using Agar dilution and Punch well diffusion method) of synthesised quinazolone derivatives bearing nalidixic acid moiety on randomly collected microbial strains. The derivatives Ga (NAH), Gb (QN) and Gd (NiQNA) showed marked inhibitory activity against enteric pathogen like Aeromonas hydrophila, a causative agent of diarrhoea in both children as well as adults. Among the respiratory pathogens included in study, derivative Gd (NiQNA) was found to be active against Streptococcus pyogenes. No significant inhibitory activity was seen by any of synthesised derivatives against Coagulase negative Staphylococcus. Derivative Ga (NAH) was found to show very high activity against the Candida colonies and derivative Gd (NiQNA) was also found to exhibit inhibitory activity against Candida albicans; a normal flora of the human body which plays an important role in causing opportunistic infections in immunocompromised hosts. Proteus vulgaris, a gram-negative bacteria included in our study was found to be inhibited by derivative Gb (QN).

Adult↗
PubMed2005

Cyclization of the substituted N-(ortho-cyclopropylphenyl)-N'-aryl ureas and thioureas in the gas phase and solution.

Electron ionization (EI), chemical ionization (CI), tandem mass spectrometry, high-resolution measurements, and labeling studies as well as quantum chemical calculations were used to understand the behavior of the molecular radical cations (EI) and protonated molecules (CI) of substituted N-(ortho-cyclopropylphenyl)-N'-aryl ureas and N-(ortho-cyclopropylphenyl)-N'-aryl thioureas in a mass spectrometer. Fragmentation schemes and possible mechanisms of primary isomerization were proposed. According to the fragmentation pattern, formation of the corresponding benzoxazines and benzothiazines was considered as the major process of isomerization of the original M(+.) and MH(+), although some portions of these ions definitely transformed into other structures. The treatment of N-(ortho-cyclopropylphenyl)-N'-phenyl urea and N-(ortho-cyclopropylphenyl)-N'-phenylthiourea in solution with strong acids formed predicted 4-ethyl-N-phenyl-4H-3,1-benzoxazin-2-amin and 4-ethyl-N-phenyl-4H-3,1-benzothiazin-2-amine as principal products.

Algorithms↗
PubMed2003

1,4-benzothiazine analogues and apoptosis: structure-activity relationship.

We have previously shown 1,4-benzothiazine (1,4-B) derivatives induce thymocyte apoptosis in vitro and thymus cell loss in vivo. Apoptosis is mediated through a complex of biochemical events including phosphatidylcholine specific-phospholipase C (PC-PLC) activation, acidic sphingomyelinase (aSMase) activation and ceramide generation, caspase-8 and caspase-3 activation. As preliminary analysis of the structure-activity relationship (SAR) suggested some structural features were responsible for apoptosis, we synthesised several derivatives and tested for apoptosis activity at equimolar concentrations. In particular, we synthesised analogues that differed in the nature of skeleton (1,4-benzothiazine, 1,4-benzoxazine and 1,2,3,4-tetrahydroquinoline) and in the nature of side chain (imidazole, benzimidazole or piperazine as azole substituent; presence, absence or transformation of alcoholic group). Results of apoptosis induction indicate that transforming the 1,4-benzothiazine skeleton into 1,2,3,4-tetrahydroquinoline does not result in significant change. Transformation into 1,4-benzoxazine decreased activity. Replacing imidazole at the side chain with different piperazines also decreased activity while replacing it with benzimidazole does not change apoptotic activity. Finally, removal of the alcoholic group by dehydration to olefin, or by transforming it into ether, increased activity. Moreover, in an attempt to analyse further the SAR characteristics that are responsible for 1,4-B-activated apoptosis we tested the effect on caspase-8,-9 and-3 activation. 1,4-B analogues activate caspases and the structural requirements correlate with those responsible for apoptosis induction.

Animals↗
PubMed2001

Cyclization of ortho-cyclopropylphenyl benzamides in gas and liquid phases.

The electron ionization (EI) and collision-induced dissociation (CID) spectra of substituted N-(ortho-cyclopropylphenyl)benzamides 1-7 and N-[ortho-(1-methylcyclopropyl)phenyl]benzamides 8-12 were recorded. In addition to routine bond cleavages, the molecular ions (M+) of 1-12 undergo cyclization into the corresponding 3-aryl-1-alkyl-1-ethyl-1H-benzoxazines and isomeric 5-ethyl-2-oxodibenzoazepines. The presence of a methyl group in the cyclopropyl ring (compounds 8-12) makes the formation of 5-ethyl-2-oxodibenzoazepine less favorable. In accord with mass spectrometric predictions, compound 13 (3-p-tolyl-1-ethyl-1H-benzoxazine) was obtained as a major product of the reaction of N-(ortho-cyclopropylphenyl)-4-methylbenzamide 1 with sulfuric acid. Traces of 5-ethyl-2-oxodibenzoazepine were also detected in gas chromatography-mass spectrometry (GC-MS) analysis of the reaction mixture although the yield was too low to allow its isolation.

Journal Article↗
PubMed1992

Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor.

Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4-methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists.

Analgesics↗
PubMed1988

Quinolone antibacterial agents. Synthesis and structure-activity relationships of 8-substituted quinoline-3-carboxylic acids and 1,8-naphthyridine-3-carboxylic acids.

A series of 7,8-disubstituted 1-cyclopropyl-6-fluoroquinoline-3-carboxylic acids, 7-substituted 1-cyclopropyl-6-fluoro-1,8-naphthyridine-3-carboxylic acids, and 10-substituted 9-fluoropyridobenzoxazine-6-carboxylic acids has been prepared and evaluated for antibacterial activity. The side chains examined at the 7-position (benzoxazine 10-position) included piperazinyl (g), 3-aminopyrrolidinyl (a), 3-(aminomethyl)pyrrolidinyl (b), and alkylated 3-(aminomethyl)pyrrolidinyl (c-f). Variations at C-8 of the quinolone ring system included hydrogen, nitro, amino, fluorine, and chlorine. The relative enhancement of in vitro activities by the side chains on the 8-hydrogen quinolone and 1,8-naphthyridine against Gram-negative organisms was a greater than b greater than g greater than c-f. The activity imparted to the substituted quinolone nucleus by the 8-substituent was in the order F greater than Cl greater than naphthyridine greater than H greater than benzoxazine greater than NH2 greater than NO2. These trends were retained in vivo.

Animals↗
PubMed2002

Highly antibacterial active aminoacyl penicillin conjugates with acylated bis-catecholate siderophores based on secondary diamino acids and related compounds.

New acylated bis-catecholates and 1,3-benzoxazine-2,4-dione derivatives based on secondary diamino acids (N-(aminoalkyl)glycines, N-aminopropyl-alanine, and N-aminopropyl-4-aminovaleric acid), on N-(aminoalkyl)aminomethyl benzoic acids, on N-(aminoalkyl)aminomethyl phenoxyacetic acids, or on 3,5-diaminobenzoic acid were synthesized as artificial siderophores. The corresponding diamino acids were obtained from the diamines and oxocarboxylic acids by catalytic hydrogenation. The acylated bis-catecholates and 1,3-benzoxazine-2,4-diones were coupled with ampicillin or amoxicillin to new siderophore aminoacylpenicillin conjugates. These conjugates exhibited very strong antibacterial activity in vitro against Gram-negative bacterial pathogens including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. The ampicillin derivative 7b (HKI 9924154) and the corresponding amoxicillin derivative 8 (HKI 9924155) represent the most active compounds. The conjugates can use bacterial iron siderophore uptake routes to penetrate the Gram-negative outer membrane permeability barrier. This was demonstrated by assays with mutants deficient in components of the iron transport systems. New siderophore penicillin V conjugates with the siderophore component attached to the phenyl ring of penicillin V are inactive against these Gram-negative bacteria.

Amino Acids↗
PubMed2000

Total synthesis of the fumiquinazoline alkaloids: solution-phase studies.

Biomimetic total syntheses of glyantrypine, fumiquinazoline F, fumiquinazoline G, and fiscalin B were achieved in four steps from tryptophan methyl ester. In the key step, the anthranilamide residue in a linear tripeptide is dehydrated to a benzoxazine by reaction with triphenylphosphine, iodine, and a tertiary amine. The benzoxazines subsequently undergo rearrangement to the natural products via an amidine intermediate. This dehydrative oxazine to quinazoline route is applicable to a broad range of N-acylanthranilamides, including sterically hindered cases.

Alkaloids↗
PubMed2003

Endothelium-independent relaxation to cannabinoids in rat-isolated mesenteric artery and role of Ca2+ influx.

(1) Three cannabinoid receptor agonists, anandamide (CB(1) receptor-selective) and the aminoalkyl-indoles, JWH 015(2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone; (CB(2) receptor-selective), R-(+)-WIN 55,212-2 (R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; slightly CB(2) receptor-selective), as well as the enantiomer S-(-)-WIN 55,212-3(S-(-)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; inactive at cannabinoid receptors), induced endothelium-independent relaxation of methoxamine-precontracted isolated small mesenteric artery of rat. KCL (60 mM) precontraction did not affect relaxation to the aminoalkylindoles, but reduced that to anandamide. (2) SR14176A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 micro M; CB(1) receptor antagonist) inhibited relaxation only to JWH 015 and anandamide. Neither AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; CB(1) antagonist) nor SR 144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; CB(2) antagonist; both at 3 micro M) affected any of the relaxations. (3) Vanilloid receptor desensitisation with capsaicin reduced anandamide relaxation; addition of SR 141716A (3 micro M) then caused further inhibition. SR 141716A did not affect capsaicin-induced relaxation. (4) The aminoalkylindoles inhibited CaCl(2)-induced contractions in methoxamine-stimulated vessels previously depleted of intracellular Ca(2+). These inhibitory effects were greatly reduced or abolished in ionomycin-(a calcium ionophore) contracted vessels. Anandamide also caused vanilloid receptor-independent, SR 141716A- (3 micro M) insensitive, inhibition of CaCl(2) contractions. (5) In conclusion, the aminoalkylindoles JWH 015, R-(+)-WIN 55,212-2 and S-(-)-WIN 55,212-3 relax rat small mesenteric artery mainly by inhibiting Ca(2+) influx into vascular smooth muscle. Anandamide causes vasorelaxation by activating vanilloid receptors, but may also inhibit Ca(2+) entry. Relaxation to JWH 015 and anandamide was sensitive to SR 141716A, but there is no other evidence for the involvement of CB(1) or CB(2) receptors in responses to these compounds.

Animals↗
PubMed2001

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

Amino Acid Sequence↗
PubMed2005

Dissecting defense-related and developmental transcriptional responses of maize during Ustilago maydis infection and subsequent tumor formation.

Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis triggers the formation of tumors on aerial parts in which the fungal life cycle is completed. A differential display screen was performed to gain insight into transcriptional changes of the host response. Some of the genes strongly up-regulated in tumors showed a pronounced developmental expression pattern with decreasing transcript levels from basal to apical shoot segments, suggesting that U. maydis has the capacity to extend the undifferentiated state of maize plants. Differentially expressed genes implicated in secondary metabolism were Bx1, involved in biosynthesis of the cyclic hydroxamic acid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one, and a novel putative sesquiterpene cyclase gene U. maydis induced (Umi)2. Together with the up-regulation of Umi11 encoding a cyclotide-like protein this suggests a nonconventional induction of plant defenses. Explicitly, U. maydis was resistant to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one but susceptible to its benzoxazolinone derivative 6-methoxy-2-benzoxazolinone. Infection studies of isolated leaves with U. maydis and Colletotrichum graminicola provided evidence for coregulation of Umi2 and PR-1 gene expression, with mRNA levels strongly determined by the extent of fungal colonization within tissue. However, in contrast to Umi2, transcript levels of PR-1 remained low in plants infected with wild-type U. maydis but were 8-fold elevated upon infection with an U. maydis mutant strongly attenuated in pathogenic development. This suggests that U. maydis colonization in planta suppresses a classical defense response. Furthermore, comparative expression analysis uncovered distinct transcriptional programs operating in the host in response to fungal infection and subsequent tumor formation.

Amino Acid Sequence↗
PubMed1989

Hydroxamic Acid glucosyltransferases from maize seedlings.

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K(m) of 174 micromolar for DIMBOA and a K(m) of 638 micromolar for DIBOA; the enzyme in peak 1 has a K(m) of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K(m). The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.

Journal Article↗
PubMed1991

Maize microsomal benzoxazinone N-monooxygenase.

The benzoxazinones occur in hydroxamic acid and lactam forms in maize (Zea mays L.) tissue. The hydroxamic acid forms which possess a N-hydroxyl group are found in the highest concentration while the lactam members which lack the N-hydroxyl group occur in lower concentrations. The hydroxamic acid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) has as its lactam counterpart 2-hydroxy-1,4-benzoxazin-3-one (HBOA). An enzyme has been identified in maize microsomal preparations which catalyzes the N-hydroxylation of HBOA to form DIBOA. The enzyme is initially observed in seedlings 2 days after imbibition which coincides with the onset of hydroxamic acid accumulation. The enzyme requires NADPH and is inhibited by sulfhydryl reagents, NADP, cytochrome c, cations, carbon monoxide, and nitrogen gas. The effect of nitrogen can be reversed by exposing the enzyme to air, while the effect of carbon monoxide can be reversed by exposing the enzyme to 450 nanometer light during the incubation period. The apparent K(m) values for HBOA and NADPH are 13 and 5 micromolar, respectively. The pH optimum is 7.5 and the temperature optimum for the enzyme is 35 degrees C. A 450 nanometer absorbance peak is observed when reduced microsomal preparations are exposed to carbon monoxide which in combination with other data presented supports the hypothesis that the enzyme is a cytochrome P-450 dependent N-monooxygenase.

Journal Article↗