Scyptolin A and B, cyclic depsipeptides from axenic cultures of Scytonema hofmanni PCC 7110.

Two novel cyclic depsipeptides were isolated from axenic cultures of the terrestrial cyanobacterium Scytonema hofmanni PCC 7110 and designated scyptolin A and B. Amino acid analyses in context with mass and 1H/13C NMR spectroscopies revealed a composition typical for heterologous cyanopeptolins but containing the uncommon residue 3'-chloro-N-methyl-Tyr (cmTyr) and a unique sidechain. Scyptolin A and B both consist of the N-acylated peptide But(1)-Ala(2)-Thr(3)-Thr(4)-Leu(5)-Ahp(6) (3-amino-6-hydroxy-2-oxo-1-piperidine)-Thr(7)-cmTyr(8)-Val(9), which forms a 19-membered ring by esterification of the carboxyl of Val(9) with the hydroxyl of Thr(4). In scyptolin B, the hydroxyl of the Thr(3) residue is additionally esterified with N-butyroyl-Ala. Both scyptolin A and B exhibit selective inhibition of porcine pancreatic elastase in vitro with IC(50) values of 3.1 microg/ml.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.     

Identification of peptide metabolites of Microcystis (Cyanobacteria) that inhibit trypsin-like activity in planktonic herbivorous Daphnia (Cladocera)

Cyanobacteria are recognized as producers of a broad variety of bioactive metabolites. Among these, the peptides synthesized by the non-ribosomal peptide synthetase pathway occur in high structural variability. One class of cyanobacterial peptides, the cyanopeptolins or micropeptins, have been shown to be strong inhibitors of vertebrate serine proteases, like trypsin. In the present study we screened extracts of ten strains of the unicellular cyanobacterium Microcystis sp. for their potential to inhibit trypsin-like activity in the planktonic crustacea Daphnia, the main herbivores in freshwater ecosystem. Respective standardized IC(50)'s varied for nearly two orders of magnitude. In HPLC fractions we could identify mainly cyanopeptolins as active compounds by MALDI-TOF mass spectrometry. Cyanopeptolins were found in 22 structural variants with 13 variants produced by one strain alone. Peptides of the microviridin class were moderately active while no activity was evident for microginins and microcystins. Among the cyanopeptolins only those were active that had an arginine or lysine residue N-terminal to the modified amino acid 3-amino-6-hydroxy-piperidone. Structural variants that had a tyrosine residue at this particular position did not inhibit trypsin-like activity. The highly variable composition of the side chain of cyanopeptolins had no marked effect on the activity. Among the six cyanobacterial strains we tested intensively two did not produce any cyanopeptolins and were accordingly less active as crude extracts. The present study underlines the potential importance of the biochemistry of cyanobacteria for the feeding ecology of a planktonic herbivore.     

Gene cloning and functional characterization of four novel antimicrobial-like peptides from scorpions of the family Vaejovidae

From the cDNA libraries made from the venom glands of two scorpions belonging to the Vaejovidae family, four different putative non disulfide-bridged antimicrobial peptides were identified: VmCT1 and VmCT2 from Vaejovis mexicanus smithi plus VsCT1 and VsCT2 from Vaejovis subcristatus. These short peptides (with only 13 amino acid residues each) share important amino acid sequence similarities among themselves and with other reported antimicrobial peptides, but their biological activities vary dramatically. This communication reports the cloning, chemical synthesis and characterization of these peptides. Two peptides, VmCT1 and VmCT2 showed broad-spectrum antibacterial activity with minimum inhibitory concentrations MICs in the range of 5-25 μM and 10-20 μM respectively, whereas their hemolytic activity at these concentrations was low. Structure-function relationships that might determine the differences in activities are discussed.     

Identification of LI-F type antibiotics and di-n-butyl phthalate produced by Paenibacillus polymyxa

Paenibacillus polymyxa strain JSa-9, a soil isolate that displayed antibacterial and antifungal activities in vitro, had been found to produce two types of antimicrobial substances. The two compounds were extracted from the fermentation broth of JSa-9 using ethyl acetate and subsequently purified by high performance liquid chromatography. By means of liquid chromatography-mass spectrometry and tandem mass spectrometry analysis, one of two antagonistic compounds was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of 883, 897, 911, 947, and 961 Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These peptides were therefore members of the LI-F group of cyclic depsipeptides.     

Potent anti-HIV activity of scytovirin domain 1 peptide

Scytovirin (SVN) is a novel anti-HIV protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. SVN contains two apparent domains, one comprising amino acids 1-48 and the second stretching from amino acids 49 to 95. These two domains display significant homology to each other and a similar pattern of disulfide bonds. Two DNA constructs encoding scytovirin 1-48 (Cys7Ser) (SD1) and 49-95 (Cys55Ser) (SD2) were constructed, and expressed in E. coli, with thioredoxin fused to their N-terminus. Purified recombinant products were tested for binding activities with the HIV surface envelope glycoproteins gp120 and gp41. Whole cell anti-HIV data showed that SD1 had similar anti-HIV activity to the full-length SVN, whereas SD2 had significantly less anti-HIV activity. Further deletion mutants of the SD1 domain (SVN(3-45)Cys7Ser, SVN(6-45)Cys7Ser, SVN(11-45)Cys7Ser) showed that the N-terminal residues are necessary for full anti-HIV activity of SD1 and that an eight amino acid deletion from the C-terminus (SVN(1-40)Cys7Ser) had a significant effect, decreasing the anti-HIV activity of SD1 by approximately five-fold.     

The depsipeptide technique applied to peptide segment condensation: scope and limitations.

A promising application of the depsipeptide technique has recently been proposed to provide ideal conditions for segment condensation, in that coupling of peptides bearing a C-terminal depsipeptide unit occurs without giving rise to epimerization at the activated amino acid. This is due to the low tendency of the activated depsipeptide units, in contrast to the corresponding peptide segments, to form optically labile oxazolones. In this work we demonstrate that coupling of depsipeptides via base-assisted activation using HBTU occurs not only without loss of configuration, but even much faster than the coupling of the corresponding all-amide segments. Nevertheless, when the coupling of long depsipeptide segments proceeds slowly, we uncovered the occurrence of beta-elimination at the activated depsipeptide unit, in an extent dependent on the presence of base in the system and on the type of the solvent. Beta-elimination was completely suppressed by using carbodiimide/HOBt activation in a non-polar solvent (DCM), and in more polar media it was limited by substituting TMP for DIEA during HBTU activation, or using particular solvent mixtures (such as DMSO/toluene) for activation via carbodiimide. Finally, we show the application of C-terminal pseudoprolines, in comparison with that of depsipeptide units, to segment coupling.     

The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors

The solution structure of the potent 95 residue anti-HIV protein scytovirin has been determined and two carbohydrate-binding sites have been identified. This unique protein, containing five structurally important disulfide bonds, demonstrates a novel fold with no elements of extended regular secondary structure. Scytovirin contains two 39 residue sequence repeats, differing in only three amino acid residues, and each repeat has primary sequence similarity to chitin binding proteins. Both sequence repeats form similarly structured domains, with the exception of one region. The result is two carbohydrate-binding sites with substantially different affinities. The unusual fold clusters aromatic residues in both sites, suggesting a binding mechanism similar to other known hevein-like carbohydrate-binding proteins but differing in carbohydrate specificity. Scytovirin, originally isolated from the cyanobacterium Scytonema varium, holds potential as an HIV entry inhibitor for both therapeutic and prophylactic anti-HIV applications. The high-resolution structural studies reported are an important initial step in unlocking the therapeutic potential of scytovirin.     

Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: toward an understanding of metabolic diversity

Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds.     

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.     

Neamphamide B, new cyclic depsipeptide, as an anti-dormant mycobacterial substance from a Japanese marine sponge of Neamphius sp

A new cyclic depsipeptide, designated neamphamide B (1), was isolated from a marine sponge of Neamphius sp. collected at Okinawa, Japan in 1993 as an anti-mycobacterial substance against active and dormant bacilli. The planar structure of neamphamide B (1) was determined on the basis of spectroscopic analysis, and stereostructure of amino acid was deduced by chromatographic comparison of the acid hydrolysate of 1 with appropriate amino acid standards after derivatizing with FDAA or GITC. Neamphamide B (1) showed potent anti-mycobacterial activity against Mycobacterium smegmatis under standard aerobic growth conditions as well as dormancy-inducing hypoxic conditions with MIC of 1.56 μg/mL. Neamphamide B (1) was also effective to Mycobacterium bovis BCG with MIC in the ranging of 6.25-12.5 μg/mL.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.     

Analysis of the temperature-sensitive mutation of Escherichia coli pantothenate kinase reveals YbjN as a possible protein stabilizer

Pantothenate kinase (PanK), a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway, catalyzes the rate-limiting phosphorylation of pantothenic acid to form phosphopantothenate during CoA biosynthesis. Escherichia coli ts9 strain manifests temperature-sensitive phenotype on LB media due to its mutation in the coaA gene (coaA1). Sequencing analysis revealed that coaA1 arises from a single base pair mutation that results in an amino acid change, L236F. This change, located proximate to the ATP binding site of CoaA, destabilizes both enzymatic activity and structural integrity or stability of the mutant protein in vitro. Spontaneously, revertants of ts9 were occasionally found on LB medium plates. Two groups of revertants were isolated: for those that can grow at 40 degrees C, a reversion of the original amino acid mutation L236F to L236L or other amino acid (such as L236C) occurs; for those that can grow at 37 degrees C but not 40 degrees C, a mutation at another gene or intergenic suppression is strongly indicated. Towards genetic identification of genes that might interact with coaA1, ybjN, which encodes a putative sensory transduction regulator protein, and whose over-expression is capable of ameliorating the temperature-sensitive phenotype of the structurally unstable CoaA1 or CoaA[L236F], was isolated. Over-expression of ybjN appears to suppress the temperature-sensitive phenotype of several other temperature-sensitive mutations, including coaA14 (carried by DV51 strain), coaA15 (carried by DV70 strain), and ilu-1, suggesting it not only helps CoaA1, but possibly works as a general stabilizer for some other unstable proteins.     

Crystal structure of a depsipeptide, Boc-(Leu-Leu-Lac)3-Leu-Leu-OEt

A sequential polydepsipeptide, Boc-(Leu-Leu-Lac)3-Leu-Leu-OEt (1) (Lac = L-lactic acid residue) has been synthesized by the segment condensation method. The sequential unit of 1, -Leu-Leu-Lac-, is consisted of two amino acid residues and one hydroxy acid residue. X-ray diffraction measurement with an imaging plate detector and a direct-methods procedure of Shake-and-Bake successfully revealed the crystal structure of 1. In the solid state, the 11-mer depsipeptide, 1, have clear alpha-helical conformation even with the three ester linkages.     

Biosynthesis of enniatin by washed cells of Fusarium sambucinum

Biosynthesis of the depsipeptide membrane ionophore--enniatin B by the washed mycelium Fusarium sambucinum Fuck 52 377 was studied. Metabolic precursors of enniatin B, alpha-ketovaleric acid, 14C-L-valine, and 14CH3-methionine, were added to the system after starvation. The amino acid content in the metabolic pool increased 1.5 times after addition of alpha-ketovaleric acid, 2.2 times after that of valine, and 2.5 times after addition of methionine. 14C-L-valine and 14CH3-methionine were incorporated into the molecule of enniatin B. Valine methylation in the molecule occurred at the level of synthesized depsipeptide. Amino acids of the metabolic pool performed the regulatory function in the synthesis.     

Molecular modeling of conformational properties of oligodepsipeptides

A depsipeptide is a chemical structure consisting of both ester and amide bonds. Quantum mechanics calculations have been performed to investigate the conformational properties of a depsidipeptide in the gas and solution phases. Similar to an alanine dipeptide, the depsidipeptide exhibits a strong preference for the polyproline II (PPII) helical conformation. Meanwhile, due to the changes in the intramolecular interaction, the propensity for beta-sheets and alpha-helices diminishes while an unusual inclination for the (phi,psi) = (-150 degrees ,0 degrees ) conformation was observed. A molecular mechanics model has been developed for polydepsipeptides based on the quantum mechanical study. Both simulated annealing and replica exchange molecular dynamics simulations have been carried out on oligodepsipeptide sequences with alternating depsi and natural residues in solution. Novel helical structures have been indicated from the simulations. When glycine is used as the alternating natural amino acid residue, the PPII conformation of a depsi residue stabilizes the peptide into a right-handed helical structure while the alpha-helical conformation of the depsi residue favors an overall left-handed helical structure. The free energy analysis indicates that both the left- and the right-handed helices are equally likely to exist. When charged lysine is introduced as the alternating natural residue, however, it is found that the depsipeptide sequence prefers an extended conformation as in PPII. Our results indicate that the depsipeptide is potentially useful in designing protein mimetics with controllable structure, function, and chemistry.     

A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium

A new anti-HIV protein, scytovirin, was isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein displayed potent anticytopathic activity against laboratory strains and primary isolates of HIV-1 with EC50 values ranging from 0.3 to 22 nM. Scytovirin binds to viral coat proteins gp120, gp160, and gp41 but not to cellular receptor CD4 or other tested proteins. This unique protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds.