The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.     

Detoxification of the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on brown mustard involves an inducible hydrolase

Brassinin is a phytoalexin produced by plants from the family Brassicaceae that displays antifungal activity against a number of pathogens of Brassica species, including Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] and L. biglobosa. The interaction of a group of isolates of L. maculans virulent on brown mustard (Brassica juncea) with brassinin was investigated. The metabolic pathway for degradation of brassinin, the substrate selectivity of the putative detoxifying hydrolase, as well as the antifungal activity of metabolites and analogs of brassinin are reported. Brassinin hydrolase activity was detectable only in cell-free homogenates resulting from cultures induced with brassinin, N'-methylbrassinin, or camalexin. The phytoalexin camalexin was a substantially stronger inhibitor of these isolates than brassinin, causing complete growth inhibition at 0.5mM.     

Phytoalexins and phytoanticipins from the wild crucifers Thellungiella halophila and Arabidopsis thaliana: rapalexin A, wasalexins and camalexin

Investigation of phytoalexin production using abiotic elicitation showed that the phytoalexin rapalexin A was produced by both Thellungiella halophila and Arabidopsis thaliana, but while A. thaliana produced camalexin, T. halophila produced wasalexins A and B and methoxybrassenin B. Considering that the genome of T. halophila is being sequenced currently and that the wasalexin pathway present in T. halophila is expected to involve a number of genes also present in Brassica species, our discovery should facilitate the isolation of genes involved in biosynthetic pathways of phytoalexins of the most economically important crucifer species.     

Camalexin induces detoxification of the phytoalexin brassinin in the plant pathogen Leptosphaeria maculans

The impact of the phytoalexins camalexin and spirobrassinin on brassinin detoxification by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], a pathogenic fungus prevalent on crucifers, was investigated. Brassinin is a plant metabolite of great significance due to its dual role both as an effective phytoalexin and as an early biosynthetic precursor of the majority of the phytoalexins produced by plants of the family Brassicaceae (Cruciferae). The rate of detoxification of brassinin in cultures of L. maculans increased substantially in the presence of camalexin, whereas spirobrassinin did not appear to have a detectable effect. In addition, the brassinin detoxifying activity of cell-free extracts obtained from cultures incubated with camalexin was substantially higher than that of control cell-free extracts or cultures incubated with spirobrassinin, and correlated positively with brassinin oxidase activity. The discovery of a potent synthetic modulator of brassinin oxidase activity, 3-phenylindole, and comparison with the commercial fungicide thiabendazole is also reported. The overall results indicate that brassinin oxidase production is induced by camalexin and 3-phenylindole but not by spirobrassinin or thiabendazole. Importantly, our work suggests that introduction of the camalexin pathway into plants that produce brassinin might make these plants more susceptible to L. maculans.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.     

Molecular cloning and biochemical characterization of Candida albicans acyl-CoA:sterol acyltransferase, a potential target of antifungal agents

To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.     

Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Extract screening by HPLC coupled to MS-MS,NMR, and CD: a dimeric and three monomeric naphthylisoquinoline alkaloids from Ancistrocladus griffithii

Three new monomeric naphthylisoquinoline alkaloids, ancistrogriffines A, B, and C, and the first dimer of a 7,8'-coupled naphthylisoquinoline, ancistrogriffithine A, have been detected by phytochemical online screening of plant extracts of Ancistrocladus griffithii, using the analytical 'triad' HPLC-MS/MS, HPLC-NMR, and HPLC-CD. Ancistrogriffithine A, as well as ancistrogriffines A and C, were structurally completely assigned (including the absolute configuration) right from the extract, without previous isolation. Furthermore, two related, but known alkaloids, ancistrocladine and hamatine, were identified. Except for ancistrogriffine B, which occurs in trace quantities only, all new alkaloids were then isolated preparatively and the initial assignments were fully confirmed by conventional offline methods. Of particular interest is the constitutionally and configurationally unprecedented structure of ancistrogriffithine A, which is simultaneously the first dimeric naphthylisoquinoline alkaloid from an Asian Ancistrocladus species. Ancistrogriffithine A and ancistrogriffine A are active against Plasmodium falciparum. Furthermore, the latter compound shows good activity against Leishmania donovani. The results demonstrate the ability of modern online methods like HPLC-NMR, -MS/MS, and -CD to serve as powerful tools for the reliable structural elucidation of even complex structures of trace compounds in crude biological matrices.     

Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis

Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 μg/ml and 16 μg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.     

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.     

Pentalenolactone biosynthesis: Molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate

Pentalenolactone (1) is an antibiotic that has been isolated from many species of Streptomyces. The putative dehydrogenase encoded by the ptlF gene (SAV2993) found within the Streptomyces avermitilis pentalenolactone gene cluster was cloned and overexpressed in Escherichia coli. PtlF, which belongs to the short-chain dehydrogenase/oxidoreductase superfamily, was shown to catalyze the oxidation of 1-deoxy-11beta-hydroxypentalenic acid (9) to 1-deoxy-11-oxopentalenic acid (10), a new intermediate of the pentalenolactone biosynthetic pathway. The methyl ester of 10 was characterized by NMR, GC-MS and high resolution mass spectrometry. PtlF exhibited a 150-fold preference for beta-NAD(+) over beta-NADP(+). PtlF had a pH optimum of 8.0 in the physiological pH range, while a significant activity enhancement was observed from pH 9.0 to 11.3. At pH 8.0, PtlF had a k(cat) of 0.65+/-0.03 s(-1), with a K(m) for 9 of 6.5+/-1.5 microM and K(m) for NAD(+) of 25+/-3 microM.     

C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-thiol proteinase activity from Paracoccidioides brasiliensis

The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulfenyl]) derivatives over thiol-containing serine proteases has never been tested. In the present work we used an extracellular serine-thiol proteinase activity from the fungal pathogen Paracoccidioides brasiliensis (PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH(2) and Bzl-MKRLTLC(Npys)-NH(2). The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L-T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH(2) (K(i)=16nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further position from the L-T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the reactivity of the C(Npys) moiety with a free SH- group.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.     

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily ω-1 and ω-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.     

Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol: Enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase

Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 °C for 9 h.     

Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.