Discovery of a Fungal Multicopper Oxidase That Catalyzes the Regioselective Coupling of a Tricyclic Naphthopyranone To Produce Atropisomers.

Atropisomeric dinapinones A1 and A2 (DPA1 and DPA2) were isolated from a culture of Talaromyces pinophilus FKI-3864. Monapinone coupling enzyme (MCE), which dimerizes naphthopyranone monapinone A (MPA), was purified from a cell-free extract of T. pinophilus FKI-3864. MCE regioselectively dimerizes MPA at the 8,8'-positions to synthesize the atropisomers DPA1 and DPA2 in a ratio of approximately 1:2.5 without a cofactor. The optimal pH value and temperature for MCE were 4.0 and 50 °C, and the apparent Km and Vmax values for MPA were (72.7±23.2) µm and (1.21±0.170) µmol min-1 mg-1 protein. The MCE polypeptide is significantly homologous with multicopper oxidases. Heterologous expression of MCE and functional analysis confirmed that MCE catalyzes the regioselective coupling reaction of MPA to produce DPA. No fungal multicopper oxidase has previously been reported to catalyze regioselective intermolecular oxidative phenol coupling to produce naphthopyranone atropisomers.

Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity.

Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

Inhibition of bacterial undecaprenyl pyrophosphate synthase by small fungal molecules.

Viridicatumtoxin and spirohexaline, small fungal molecules with a tetracyclic scaffold and an additional spirobicyclic ring in common, were found to inhibit bacterial undecaprenyl pyrophosphate (UPP) synthase with IC50 values of 4 and 9 µm, respectively. These molecules showed weak inhibitory activity against catalytically related enzymes such as bacterial octaprenyl pyrophosphate synthase and yeast dehydrodolichyl pyrophosphate synthase, indicating that the compounds preferentially inhibit UPP synthase. They showed antimicrobial activity, particularly against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, molecular modeling strongly suggested that the hydrophobic spirobicyclic ring of viridicatumtoxin interacts with three hydrophobic clefts of the active site in MRSA UPP synthase.

Epi-trichosetin, a new undecaprenyl pyrophosphate synthase inhibitor, produced by Fusarium oxysporum FKI-4553.

A new compound, designated epi-trichosetin (1), was isolated along with the known compound trichosetin (2) from the culture broth of Fusarium oxysporum FKI-4553 by solvent extraction, silica gel column chromatography and reversed-phase HPLC. The structure of 1 was elucidated by comparing various spectral data with those of 2, revealing that 1 was a stereoisomer of 2. Compounds 1 and 2 inhibited the undecaprenyl pyrophosphate synthase activity of Staphylococcus aureus with IC50 values of 83 and 30 µM, respectively, and showed antimicrobial activity, particularly against Gram-positive bacteria, including methicillin-sensitive and -resistant S. aureus.

Spirohexalines, new inhibitors of bacterial undecaprenyl pyrophosphate synthase, produced by Penicillium brasilianum FKI-3368.

An enzyme assay for bacterial undecaprenyl pyrophosphate (UPP) synthase was performed to screen microbial culture broths for inhibitors of UPP synthase. During the course of this screening program, an EtOH extract of a rice culture of Penicillium brasilianum FKI-3368 was found to inhibit UPP synthase activity. From activity-guided purification, a new compound-designated spirohexaline was isolated together with the structurally related and known viridicatumtoxin by ethyl acetate extraction silica gel and octadecylsilane column chromatographies and high-performance liquid chromatography. The structure of spirohexaline was elucidated by spectroscopic analysis, including NMR. Spirohexaline and viridicatumtoxin have a common hexacycline structure produced by fusion of a tetracycline-type ring with a spiro-type ring. They inhibited UPP synthase activity with IC50 values of 9.0 and 4.0 µM, respectively.

Anti-infectious agents against MRSA.

Clinically useful antibiotics, ß-lactams and vancomycin, are known to inhibit bacterial cell wall peptidoglycan synthesis. Methicillin-resistant Staphylococcus aureus (MRSA) has a unique cell wall structure consisting of peptidoglycan and wall teichoic acid. In recent years, new anti-infectious agents (spirohexaline, tripropeptin C, DMPI, CDFI, cyslabdan, 1835F03, and BPH-652) targeting MRSA cell wall biosynthesis have been discovered using unique screening methods. These agents were found to inhibit important enzymes involved in cell wall biosynthesis such as undecaprenyl pyrophosphate (UPP) synthase, FemA, flippase, or UPP phosphatase. In this review, the discovery, the mechanism of action, and the future of these anti-infectious agents are described.

Molecular cloning of the gene cluster for lariatin biosynthesis of Rhodococcus jostii K01-B0171.

The biosynthetic gene cluster for lariatins A and B, anti-mycobacterial peptide antibiotics with a unique "lasso" structure, was cloned from Gram-positive bacterium Rhodococcus jostii K01-B0171. Random transposition mutagenesis using IS1415 derivative was carried out to identify a chromosomal locus involved in lariatin biosynthesis and six independent lariatin non-producing variants were obtained. Arbitrary PCR revealed that one insertion was located near the region involved in lariatin biosynthesis. Using the lariatin gene as a probe, a genomic library of R. jostii K01-B0171 was screened by colony hybridization, and two clones were obtained. Sequence analysis of these clones revealed that the gene cluster for lariatin biosynthesis spanning about 4.5 kb consisted of five open reading frames (larA to larE). We proposed that the linear precursor LarA is processed by LarB, LarC, and LarD, and the mature lariatin is exported by LarE.

The high mannose-type glycan binding lectin actinohivin: dimerization greatly improves anti-HIV activity.

The actinomycete-derived lectin actinohivin (AH) inhibits entry of HIV-1 to susceptible cells at low nM concentrations. The cooperative binding of three segments of AH to three high mannose-type glycans (HMTGs) of HIV-1 gp120 generates specific and strong anti-HIV activity. Dimerization of AH effectively improves anti-HIV activity by increasing the number of HMTG-binding pockets. AH dimers were prepared using an Escherichia coli expression system and their anti-syncytium formation and anti-HIV activities were evaluated. Each dimer was constructed by a head-to-tail fusion of two AH molecules, with or without a spacer. As a result, His-TEV-AH/RTB(132-143)/AH, which has the residues 132-143 of ricin toxin B-chain (RTB) as a spacer, had 20-fold higher anti-syncytium formation activity and also exhibited 2-30-fold higher anti-HIV activity than AH against various clinically isolated HIV-1 strains, including drug-resistant ones. Mutation analysis implies that all six HMTG-binding pockets of the dimer participated in HMTG binding. Several AH dimers with different spacer sequences showed diverse activities, suggesting that the spacer sequence is an important factor to create higher anti-HIV activity. A dimer with improved anti-HIV activity would be a good candidate for investigation as a potential microbicide to prevent HIV transmission.

Synthesis of (-)-homogalanthamine from naltrexone.

Acetylcholinesterase inhibitor (-)-homogalanthamine 3 was synthesized from µ opioid antagonist naltrexone (2) in 16% total yield. The synthesis features Grob fragmentation as a key reaction, which was especially accelerated in the presence of 15-crown-5.

Actinohivin: specific amino acid residues essential for anti-HIV activity.

Actinohivin (AH) is a microbial lectin containing 114 amino acids, which inhibits human immunodeficiency virus (HIV) infection. This effect is brought about by its specific binding to Man-α(1-2)-Man unit(s) of high-mannose type glycan (HMTG) bound to HIV gp120. The recently determined crystal structure of AH suggests that three repeated segments (the residue numbers 1-38, 39-76 and 77-114 for segments 1, 2 and 3, respectively) form three sugar-binding pockets to accommodate Man-α(1-2)-Man units. The strong specific binding of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the 'cluster effect' of lectin. It remains to be seen which residues of the sugar-binding pockets are essential for acceptance of Man-α(1-2)-Man. To identify the amino acid residues critical for anti-HIV effect, we performed mutational analysis. Mutant AHs were subjected to enzyme-linked immunosorbent assay testing for gp120-binding activity and to syncytium formation assay. As a result, it was revealed that Asp15, Tyr23, Leu25, Asn28 and Tyr32 in segment 1, Tyr61 in segment 2 and Tyr99 in segment 3 are essential for anti-HIV activity. The conserved residues, Asp53, Leu63, Asn66 and Tyr70, in segment 2 and, Asp91, Leu101, Asn104 and Tyr108, in segment 3 are also necessary. Furthermore, aromatic residues at positions 23 and 32 are required for creation of potency. These data will be useful for predicting the detailed mechanism of AH-Man-α(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.

Production of a new type of amidepsine with a sugar moiety by static fermentation of Humicola sp. FO-2942.

Static fermentation of amidepsine-producing fungus Humicola sp. FO-2942 led to the production of six new amidepsines, including a new type of glycosylated congener. Non-glycosylated amidepsine J inhibited both human diacylglycerol acyltransferases 1 (DGAT1) and DGAT2 with the same IC(50) value of 40 muM, whereas glycosylated amidepsines F to I showed very weak inhibitory activity against DGAT1 and DGAT2.

Mechanism by which the lectin actinohivin blocks HIV infection of target cells.

Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man alpha(1-2)Man, Man alpha(1-2)Man alpha(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K(d) = 3.4 x 10(-8) M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the "cluster effect" of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.

Expression of two human acyl-CoA:diacylglycerol acyltransferase isozymes in yeast and selectivity of microbial inhibitors toward the isozymes.

Two isozymes for human acyl-coenzyme A:diacylglycerol acyltransferase (DGAT), DGAT1 and DGAT2, were independently expressed in DGAT-deficient Saccharomyces cerevisiae to establish DGAT1- and DGAT2-S. cerevisiae. The selectivity of DGAT inhibitors of natural origin towards the isozymes was assessed in enzyme assays using the microsomal fractions prepared from DGAT1- and DGAT2-S. cerevisiae. Amidepsines and xanthohumol inhibited DGAT1 and DGAT2 with similar potency, whereas roselipins were found to inhibit DGAT2 selectively.

Lariatins, novel anti-mycobacterial peptides with a lasso structure, produced by Rhodococcus jostii K01-B0171.

Two anti-mycobacterial peptides with a lasso structure, named lariatins A and B, were separated by HP-20 and ODS column chromatographies and purified by HPLC from the culture broth of Rhodococcus jostii K01-B0171, which was isolated from soil aggregates collected in Yunnan, China. Lariains A and B showed growth inhibition against Mycobacterium smegmatis with MIC values of 3.13 and 6.25 microg/ml in agar dilution method, respectively. Furthermore, lariatin A inhibited the growth of Mycobacterium tuberculosis with an MIC of 0.39 microg/ml in liquid microdilution method.

Essential regions for antiviral activities of actinohivin, a sugar-binding anti-human immunodeficiency virus protein from an actinomycete.

Actinohivin (AH) is a potent anti-human immunodeficiency virus (HIV) protein that consists of highly conserved three-tandem repeats (segments 1, 2, and 3). The molecular target of AH in its anti-HIV activity is high-mannose-type saccharide chains of HIV gp120. This article deals with sequence requirements for the anti-HIV activity of AH. The deleted or substituted DNAs encoding AH or His-AH were prepared using mutagenic oligonucleotide primers in PCR. The mutant constructs were expressed in Escherichia coli, and the activities of the recombinant protein products were examined by a syncytium-formation assay system that mimics anti-HIV activity. The single segment mutant His-AHs showed no anti-syncytium-formation activity, but the mutant His-AHs, which consists of 2 or 3 segments, retained reduced activities. His-AH(6-114) dramatically reduced the anti-syncytium-formation activity to that of His-AH(36-114) or His-AH(I5A). Furthermore, His-AH(Q33A), His-AH(Q71A), and His-AH(Q109A) in which glutamine residues were substituted into alanine showed reduced activities of 1/20, 1/10, and 1/30, respectively, in anti-syncytium formation compared with His-AH. These results indicate that three segments of AH are necessary for potent anti-syncytium-formation activity-that is, for potent anti-HIV activity and the cooperated involvement of each segment of AH increased the AH-gp120 interaction.

Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120.

We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH-gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC50 = 3.0 microg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.