Hydrophobic derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate: a new type of D-glucosamine-6-phosphate synthase inhibitors with antifungal action

Several N-acyl and ester derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) have been synthesised and tested as inhibitors of fungal enzymes involved in early steps of chitin biosynthesis and for antifungal activity. All the tested derivatives were found to be much poorer inhibitors of the enzyme, D-glucosamine-6-phosphate (GlcN-6-P) synthase, than the parent compound but some of them exhibited much better antifungal activity. MIC values for the investigated compounds ranged between 10 mg mL(-1), found for ADGP and 0.3 mg mL(-1) for the most active derivative, namely ADGP dimethyl ester. Increased affinity of ADGP derivatives to the artificial immobilised cell membrane was correlated with their enhanced ability to be taken up by fungal cells by free diffusion. It was found that some of the examined derivatives behaved as 'pro-drugs' and after internalisation were converted into ADGP in the cell-free extract. This conversion was relatively rapid for ADGP esters but very slow for N-acyl derivatives. Results of our studies demonstrate a possibility of design and preparation of GlcN-6-P synthase inhibitors exhibiting antifungal activity.     

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.     

Analysis of the Diluents Used for Rubella Virus Hemagglutination and Hemagglutination-Inhibition Tests

A new diluent (ADGP) for the rubella virus hemagglutination (HA) and hemagglutination-inhibition (HI) tests is described. It was found that the HA and HI titers and the erythrocyte agglutination pattern were improved in ADGP compared to previously described diluents. The influence of the components of ADGP and of various test conditions on optimal HA and HI results were examined.     

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.     

Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.     

Effect of biocontrol agent Pseudomonas fluorescens 2P24 on soil fungal community in cucumber rhizosphere using T-RFLP and DGGE

Fungi and fungal community play important roles in the soil ecosystem, and the diversity of fungal community could act as natural antagonists of various plant pathogens. Biological control is a promising method to protect plants as chemical pesticides may cause environment pollution. Pseudomonas fluorescens 2P24 had strong inhibitory on Rastonia solanacearum, Fusarium oxysporum and Rhizoctonia solani, etc., and was isolated from the wheat rhizosphere take-all decline soils in Shandong province, China. However, its potential effect on soil fungal community was still unknown. In this study, the gfp-labeled P. fluorescens 2P24 was inoculated into cucumber rhizosphere, and the survival of 2P24 was monitored weekly. The amount decreased from 10(8) to 10(5) CFU/g dry soils. The effect of 2P24 on soil fungal community in cucumber rhizosphere was investigated using T-RFLP and DGGE. In T-RFLP analysis, principle component analysis showed that the soil fungal community was greatly influenced at first, digested with restriction enzyme Hinf I and Taq I. However, there was little difference as digested by different enzymes. DGGE results demonstrated that the soil fungal community was greatly shocked at the beginning, but it recovered slowly with the decline of P. fluorescens 2P24. Four weeks later, there was little difference between the treatment and control. Generally speaking, the effect of P. fluorescens 2P24 on soil fungal community in cucumber rhizosphere was just transient.     

The effect of acid irrigation on enzyme activities of the single partners of ectomycorrhizas from a limed stand of Norway spruce (Picea abies [L.] Karst.)

The aim of our investigations was to elucidate the effects of acid precipitation on some enzymes of the primary metabolism of ectomycorrhizas. Mycorrhizas of the type of Piceirhiza nigra Gronbach and of Russula ochroleuca (Pers.) Fr. and Tuber puberulum Berk. and Br. were collected from a stand of Norway spruce (Picea abies [L.] Karst.) during the growing seasons of 1991 and 1992. The experimental plots had been limed (Ca: 22 kmol ha_-1, Mg: 20 kmol ha_-1) in 1984 and exposed to acid irrigation (pH 2.7–2.8, H₂SO₄: 2 kmol ha_-1 a_-1) from 1984 to 1990. Crude extracts of mycorrhizas were assayed for the activities of glucose-6-phosphate dehydrogenase (G6P-DH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). The influence of the experimental treatments on these enzyme activities of the primary metabolism was generally low. For P. nigra, the activity of G6P-DH was decreased on the irrigated plot (photometric determinations). This seems to be a selective effect on the fungal partner, since quantitative enzyme electrophoresis revealed a decrease in the percentage of the fungal enzyme activity in relation to the total enzyme activity, whereas the content of the fungal compound ergosterol was not affected. A decrease in the fungal G6P-DH activity could also be detected in mycorrhizas of Tuber puberulum. There was also a seasonal variation in the proportion of fungal activity of G6P-DH in relation to the total G6P-DH activity. In the photometric assay (total activity) the effect was not discernible. This is indicative of a degree of regulation between the two partners, which could only be detected by quantitative enzyme electrophoresis. In addition, it could be deduced from the electrophoretograms, that in the case of G6P-DH and 6PG-DH the fungal enzyme activity was dominating in all mycorrhizas studied whereas in the case of ICA-DH the fungal band varied from being conspicuous to absent in different species of mycorrhizas. The banding pattern of G6P-DH was reproducibly different for all investigated species of mycorrhizas.     

Functional diversity of cytochrome P450s of the white-rot fungus Phanerochaete chrysosporium

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions.     

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.     

Mariannaepyrone--a new inhibitor of thromboxane A2 induced platelet aggregation

Mariannaeapyrone ((E)-2-(1,3,5,7-tetramethyl-5-nonenyl)-3,5-dimethyl-6-hydroxy-4H-pyran-4-one) is a new fungal metabolite isolated from fermentations of the common mycophilic deuteromycete Mariannaea elegans. The chemical structure of the 4-pyrone was determined by spectroscopic techniques. Mariannaeapyrone is a selective inhibitor of the thromboxane A2 induced aggregation of human platelets, whereas only weak cytotoxic and antimicrobial effects could be observed.     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Synthesis of some thiazolyl aminobenzothiazole derivatives as potential antibacterial, antifungal and anthelmintic agents

A series of 4-(6-substituted-1,3-benzothiazol-2-yl)amino-2-(4-substitutedphenyl)- amino-1,3-thiazoles, 9-24 have been synthesised from 2-chloro-N-(6-substituted-1,3-benzothiazol-2-yl)acetamides, 5-8. The structures of these compounds have been elucidated by spectral (IR, (1)H NMR, Mass) and elemental (C, H, N) analysis data. All the newly synthesised compounds (9-24) were screened for their antibacterial, antifungal and anthelmintic activities. Almost all of these compounds showed moderate to good antimicrobial activity against two gram negative bacteria (E. coli, P. aeruginosa), two gram positive bacteria (S. aureus, B. subtilis), pathogenic fungal strains (C. albicans, A. niger) and good anthelmintic activity against earthworm species (P. corethruses). Compounds 18 and 20 exhibited good antibacterial and antifungal activities, while compound 22 displayed the most significant anthelmintic activity.     

Influence of Pleurotus ostreatus inoculation on wood degradation and fungal colonization

The influence of Pleurotus ostreatus inoculation on wood degradation and on fungal community structure was studied. The experiments were performed on an organically poor fly ash deposit covered with a 10 cm layer of beech wood chips inoculated with P. ostreatus isolate ZIM76. Compared to non-inoculated wood chips, inoculation increased the temperatures and relative humidities and, in the first 6 months, accelerated Klason lignin degradation by 9% and also, after 17 months, increased iron translocation into wood chips by 30%. After 6 months, PCR-DGGE showed 22-28 and 13-21 fungal taxa in non-inoculated and P. ostreatus-inoculated beech chips, respectively. The differences in number of taxa and in the fungal community structure (based on Dice coefficient) between non-inoculated and inoculated wood chips diminished with time. The results indicate that the naturally occurring processes of wood degradation are as efficient as those occurring in sites inoculated with P. ostreatus.     

Differential responses of lichen symbionts to enhanced nitrogen and phosphorus availability: an experiment with Cladina stellaris

BACKGROUND AND AIMS: Lichens can be both nitrogen- (N) and phosphorous- (P) limited and thus may be susceptible to nutrient enrichment. Nutrient enrichment with N and P may have differing impacts on the lichen structure because of different physiological responses of fungal and algal partners to these nutrients. The hypothesis was tested that the differential responses of lichen symbionts to enhanced availability of N and P is reflected in the lichen thallus structure and the wall-to-wall interface between the algal and fungal cells. METHODS: Lichen cushions of Cladonia stellaris were treated with one P and two N concentrations alone and in combination that yielded total depositions of approx. 300 (moderate) and 1000 (high) mg N m(-2) and 100 (high) mg P m(-2) over an experiment lasting 14 weeks. The effects of N and P inputs on the relative volumes of fungal and algal cell in the medullary tissue and on the thallus structure were studied using light microscopy. The interface between algal and fungal cell walls was examined using transmission electron microscopy. KEY RESULTS: The influence of excess P on the lichen thallus structure was stronger than that of additional N. Addition of P reduced the N : P ratio in podetia, the proportion of the medullary layer volume occupied by the algal cells, the thallus volume occupied by the internal lumen, and the algal cell-wall area covered by fungal hyphae. CONCLUSIONS: Ecologically realistic changes in the availability of key macronutrients can alter the growth of symbionts. Reduction in the proportion of photobiont cells indicates that the application of P either stimulates fungal hyphal growth in the medullary tissue or impairs the cell division of the algal cells. The results suggest that both the N and P availability and thallus N : P ratio affect the growth rates of lichen symbionts.     

Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5.

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.     

Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis based on rDNA sequences.

The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified from total DNA extracted from frond tissues of Livistona chinensis with universal and fungal-specific primers. These amplified fragments were cloned and sequenced. Phylogenetic analysis based on the 5.8S gene sequences indicated that the six clone sequences obtained were of different origins. Five sequences, P1-9, P2-6, P4-4, P4-5, and P4-7, belonged to the fungi and one sequence, P3-2, belonged to the plants. P1-9 was inferred to belong to the Basidiomycota based on the phylogenetic analysis of the 5.8S gene sequences but could not be identified to lower taxonomic levels. Further identification of the other four fungal clones to lower taxonomic levels was attempted based on phylogenetic analysis and sequence comparison of both the conserved 5.8S gene and the variable ITS regions. The origin of P2-6 was identified to be Glomerella and its anamorph Colletotrichum, the origins of P4-5 and P4-7 were Mycosphaerella and its anamorph Cladosporium, and the origin of P4-4 was the Herpotrichiellaceae. The direct approach to detection and taxonomic placement of endophytic fungi within host tissue without the need for conventional in vitro culturing is discussed.     

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (?4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.