Isolation and structure determination of new linear azole-containing peptides spongiicolazolicins A and B from Streptomyces sp. CWH03.

Linear azole-containing peptides are a class of ribosomally synthesized and post-translationally modified peptides. We performed a chemical investigation on marine actinomycetes, and new linear azole-containing peptides named spongiicolazolicins A and B were found in the MeOH extracts of a newly isolated strain Streptomyces sp. CWH03 (NBRC 114659) and two strains of S. spongiicola (strain HNM0071T: DSM 103383T and strain 531S: NBRC 113560). The strain Streptomyces sp. CWH03 was indicated to be a new species closely related to S. spongiicola by phylogenetic analysis using the genome sequence. The new peptides named spongiicolazolicins A and B were isolated from the cell of Streptomyces sp. CWH03. The partial structure of spongiicolazolicin A was determined by 2D NMR experiments. Based on data of MS/MS experiments, the chemical structures of spongiicolazolicins A and B were proposed using the amino acid sequence deduced from the precursor-encoding gene, which was found from whole-genome sequence data of Streptomyces sp. CWH03. The biosynthetic gene cluster of spongiicolazolicins was proposed based on comparative analysis with that of a known linear azole peptide goadsporin. KEY POINTS: • Streptomyces sp. CWH03 was a new species isolated from marine sediment. • New linear azole-containing peptides named spongiicolazolicins A and B were isolated. • Biosynthetic pathway of spongiicolazolicins was proposed.

Structures and Catalytic Activities of Complexes between Heme and All Parallel-Stranded Monomeric G-Quadruplex DNAs.

Heme in its ferrous and ferric states [heme(Fe2+) and heme(Fe3+), respectively] binds selectively to the 3'-terminal G-quartet of all parallel-stranded monomeric G-quadruplex DNAs formed from inosine(I)-containing sequences, i.e., d(TAGGGTGGGTTGGGTGIG) DNA(18mer) and d(TAGGGTGGGTTGGGTGIGA) DNA(18mer/A), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form 1:1 complexes [heme-DNA(18mer) and heme-DNA(18mer/A) complexes, respectively]. These complexes exhibited enhanced peroxidase activities, compared with that of heme(Fe3+) alone, and the activity of the heme(Fe3+)-DNA(18mer/A) complex was greater than that of the heme(Fe3+)-DNA(18mer) one, indicating that the 3'-terminal A of the DNA sequence acts as an acid-base catalyst that promotes the catalytic reaction. In the complexes, a water molecule (H2O) at the interface between the heme and G-quartet is coordinated to the heme Fe atom as an axial ligand and possibly acts as an electron-donating ligand that promotes heterolytic peroxide bond cleavage of hydrogen peroxide bound to the heme Fe atom, trans to the H2O, for the generation of an active species. The intermolecular nuclear Overhauser effects observed among heme, DNA, and Fe-bound H2O indicated that the H2O rotates about the H2O-Fe coordination bond with respect to both the heme and DNA in the complex. Thus, the H2O in the complex is unique in terms of not only its electronic properties but also its dynamic ones. These findings provide novel insights into the design of heme-deoxyribozymes and -ribozymes.

Isolation and structure determination of a new lasso peptide specialicin based on genome mining.

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.

Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranea.

A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.

Isolation and structure determination of a new lantibiotic cinnamycin B from Actinomadura atramentaria based on genome mining.

New lantibiotic cinnamycin B was isolated from the extract of Actinomadura atramentaria NBRC 14695(T), based on genome mining and chemical investigation. The partial structure of cinnamycin B was established by 2D NMR experiments, which indicated that cinnamycin B had same methyl lanthionine bridging pattern with cinnamycin. The reduction with NaBH4-NiCl2 afforded the reduced cinnamycin B, and MS/MS experiment indicated the presence of hydroxy asparatic acid in the molecule. Cinnamycin B showed an antibacterial activity against Streptomyces antibioticus with dosage of 5 µg (0.5µL, 10 mg/mL solution) at spot-on-lawn testing method. The gene cluster of cinnamycin B on the genome of A. atramentaria was identified and discussed in comparison with that of cinnamycin.

Structure determination of a siderophore peucechelin from Streptomyces peucetius.

Previously, Park et al. isolated a new siderophore from Streptomyces peucetius ATCC 27952 based on information of the genome sequence and the structure of the siderophore was deduced to be a cyclic peptide based on MS/MS analysis. To clarify the structure of the siderophore, we cultured S. peucetius with iron deficient medium. Through several chromatographic procedures, the siderophore named peucechelin was isolated with the yield enough to perform NMR experiments. The planar structure of peucechelin was elucidated by the combination of ESI-MS experiment and NMR spectroscopic analyses of the gallium (III) complex. Unlike the previously deduced cyclic structure, the structure was determined to be a linear peptide which was similar to a known siderophore foroxymithine. The stereochemistries of amino acids constituting peucechelin were determined by applying modified Marfey method to the hydrolysate. Since the biosynthetic gene of peucechelin was formerly determined by Park et al. the similar genes were searched using genome data of other streptomycetes. As a result, the similar genes were found in the genome data of S. venezuelae and S. purpureus. Isolation and identification of siderophore was performed from the iron deficient culture of S. venezuelae. The siderophore of S. venezuelae was identified to be known compound foroxymithine by analysis ESI-MS and NMR spectra in the similar manner with peucechelin. Production of foroxymithine was also observed in the iron deficient culture of S. purpureus. Based on the genome data, comparison of the biosynthetic genes of structurally related siderophores peucechelin and foroxymithine was accomplished in discussion.

Isolation and structure determination of new siderophore albachelin from Amycolatopsis alba.

A new siderophore named albachelin was isolated from iron deficient culture of Amycolatopsis alba. The planar structure of albachelin was elucidated by the combination of ESI-MS/MS experiment and NMR spectroscopic analyses of the gallium (III) complex. The structure of albachelin was determined to be a linear peptide consisting of 6 mol of amino acids including 3 mol of serine, one mol each of N-α-acethyl-N-δ-hydroxy-N-δ-formylornithine, N-α-methyl-N-δ-hydroxyornithine, and cyclic N-hydroxyornithine. The stereochemistries of amino acids constituting albachelin were analyzed by applying modified Marfey method to the hydrolysate of albachelin. Based on bioinformatics, we deduced and discussed the possible biosynthetic gene cluster involved in albachelin biosynthesis from the genome sequence of A. alba. By prediction of substrates for adenylation domains, a non-ribosomal peptide biosynthetase gene (AMYAL_RS0130210) was proposed to be the main biosynthetic gene for albachelin biosynthesis. The related genes including transporter for siderophore were found near the NRPS gene as a gene cluster.

[A Case of Bacterial Meningitis in Which Occurrence of Ataxie Optique Led to Detection of a Small Subdural Abscess and Pialitis].

We report the case of a 69-year-old man with bacterial meningitis who presented with ataxie optique in the peripheral part of the left visual field in both hands. A detailed neurological examination with contrast-enhanced brain MRI in the early stage of the clinical course identified a small subdural abscess and pialitis in the right parietal area. A favorable outcome was obtained with antibiotic therapy alone. In a case with higher brain dysfunction of unknown cause in the clinical course of bacterial meningitis, a detailed neurological examination may be helpful to identify the causative site. (Received September 25, 2023; Accepted October 31, 2023; Published March 1, 2024).

Inversion of the stereochemistry around the sulfur atom of the axial methionine side chain through alteration of amino acid side chain packing in Hydrogenobacter thermophilus cytochrome C552 and its functional consequences.

In cytochrome c, the coordination of the axial Met Sδ atom to the heme Fe atom occurs in one of two distinctly different stereochemical manners, i.e., R and S configurations, depending upon which of the two lone pairs of the Sδ atom is involved in the bond; hence, the Fe-coordinated Sδ atom becomes a chiral center. In this study, we demonstrated that an alteration of amino acid side chain packing induced by the mutation of a single amino acid residue, i.e., the A73V mutation, in Hydrogenobacter thermophilus cytochrome c552 (HT) forces the inversion of the stereochemistry around the Sδ atom from the R configuration [Travaglini-Allocatelli, C., et al. (2005) J. Biol. Chem. 280, 25729-25734] to the S configuration. Functional comparison between the wild-type HT and the A73V mutant possessing the R and S configurations as to the stereochemistry around the Sδ atom, respectively, demonstrated that the redox potential (Em) of the mutant at pH 6.00 and 25 °C exhibited a positive shift of ∼20 mV relative to that of the wild-type HT, i.e., 245 mV, in an entropic manner. Because these two proteins have similar enthalpically stabilizing interactions, the difference in the entropic contribution to the Em value between them is likely to be due to the effect of the conformational alteration of the axial Met side chain associated with the inversion of the stereochemistry around the Sδ atom due to the effect of mutation on the internal mobility of the loop bearing the axial Met. Thus, the present study demonstrated that the internal mobility of the loop bearing the axial Met, relevant to entropic control of the redox function of the protein, is affected quite sensitively by the contextual stereochemical packing of amino acid side chains in the proximity of the axial Met.

Interaction between the heme and a G-quartet in a heme-DNA complex.

The structure of a complex between heme(Fe(3+)) and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized by (1)H NMR. The study demonstrated that the heme(Fe(3+)) is sandwiched between the 3'-terminal G-quartets of the G-quadruplex DNA. Hence, the net +1 charge of the heme(Fe(3+)) in the complex is surrounded by the eight carbonyl oxygen atoms of the G-quartets. Interaction between the heme Fe(3+) and G-quartets in the complex was clearly manifested in the solvent (1)H/(2)H isotope effect on the NMR parameters of paramagnetically shifted heme methyl proton signals, and interaction of the heme Fe(3+) with the eight carbonyl oxygen atoms of the two G-quartets was shown to provide a strong and axially symmetric ligand field surrounding the heme Fe(3+), yielding a heme(Fe(3+)) low-spin species with a highly symmetric heme electronic structure. This finding provides new insights as to the design of the molecular architecture and functional properties of various heme-DNA complexes.

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys¹4-Cys³9 bond into the ovomucoid third domain from silver pheasant.

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹4-Cys³9 bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹4-Cys³9 bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.

Isolation and structure determination of new chymotrypsin inhibitory peptides streptopeptolins B and C.

New chymotrypsin inhibitory peptides named streptopeptolins B and C were isolated from Streptomyces olivochromogenes. Structures of streptopeptolins B and C were determined to be cyclic depsipeptides possessing 3-amino-6-hydroxy-2-piperidone unit by interpretation of NMR spectra and ESI-MS. Streptopeptolins B and C showed inhibitory activities to chymotrypsin with IC50 of 8.0 and 12.0 µg/mL, respectively.

Heterologous production of new lasso peptide koreensin based on genome mining.

Lasso peptides are a class of ribosomally biosynthesized and posttranslationally modified peptides with a knot structure as a common motif. Based on a genome search, a new biosynthetic gene cluster of lasso peptide was found in the genome of the proteobacterium Sphingomonas koreensis. Interestingly, the amino acid sequence of the precursor peptide gene includes two cell adhesion motif sequences (KGD and DGR). Heterologous production of the new lasso peptide was performed using the cryptic biosynthetic gene cluster of S. koreensis. As a result, a new lasso peptide named koreensin was produced by the gene expression system in the host strain Sphingomonas subterranea. The structure of koreensin was determined by NMR and ESI-MS analysis. The three-dimensional structure of koreensin was obtained based on an NOE experiment and the coupling constants. A variant peptide (koreensin-RGD), which had RGD instead of KGD, was produced by heterologous production with site-directed mutagenesis experiment. Koreensin and koreensin-RGD did not show cell adhesion inhibitory activity, although the molecules possessed cell adhesion motifs. The possible presence of a salt bridge between the motifs in koreensin was indicated, and it may prevent the cell adhesion motif from functioning.

Isolation and structure determination of a new antibacterial peptide pentaminomycin C from Streptomyces cacaoi subsp. cacaoi.

A new antibacterial peptide named pentaminomycin C was isolated from an extract of Streptomyces cacaoi subsp. cacaoi NBRC 12748T, along with a known peptide BE-18257A. Pentaminomycin C was determined to be a cyclic pentapeptide containing an unusual amino acid, Nδ-hydroxyarginine (5-OHArg), by a combination of ESI-MS and NMR analyses. The structure of pentaminomycin C was determined to be cyclo(-L-Leu-D-Val-L-Trp-L-5-OHArg-D-Phe-). Pentaminomycin C exhibited antibacterial activities against Gram-positive bacteria including Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus. The biosynthetic gene cluster for pentaminomycin C and BE-18257A was identified from the genome sequence data of S. cacaoi subsp. cacaoi.

Heterologous production of coryneazolicin in Escherichia coli.

Coryneazolicin is a plantazolicin family peptide, belonging to linear azole-containing peptides (LAPs). Although coryneazolicin was previously synthesized by in vitro experiments, its biological activity has not been evaluated. In this report, the heterologous production of coryneazolicin was accomplished to obtain enough coryneazolicin for biological activity tests. The structure of coryneazolicin was confirmed by ESI-MS and NMR analyses. The biological activity tests indicated that coryneazolicin possessed potent antibacterial activity and cytotoxicity. Although antibacterial activity of plantazolicin was previously reported, cytotoxicity was newly found in coryneazolicin among plantazolicin type peptides. In addition, we revealed that coryneazolicin induced apoptosis on HCT116 and HOS cancer cell lines.

Isolation and structure determination of a new cytotoxic peptide, curacozole, from Streptomyces curacoi based on genome mining.

Using genome mining, a new cytotoxic peptide named curacozole was isolated from Streptomyces curacoi. Through ESI-MS and NMR analyses, curacozole was determined to be a macrocyclic peptide containing two isoleucine, two thiazole and three oxazole moieties. Curacozole exhibited potent cytotoxic activity against HCT116 and HOS cancer cells. The proposed biosynthetic gene cluster of curacozole was identified and compared with that of the related compound YM-216391.

Streptopeptolin, a Cyanopeptolin-Type Peptide from Streptomyces olivochromogenes.

Cyanopeptolin-type peptides are cyclic depsipeptides that commonly have 3-amino-6-hydroxy-2-piperidone (Ahp) unit in the molecules. So far, cyanopeptolin-type peptides have been isolated as protease inhibitors from a wide variety of cyanobacteria. In the course of screening for new peptides, a new peptide streptopeptolin, which had the similar structure to cyanopeptolin, was isolated from the extract of Streptomyces olivochromogenes NBRC 3561. Streptopeptolin is the first cyanopeptolin-type peptide isolated from actinobacteria. The structure of streptopeptolin was determined by the analysis of electrospray ionization mass spectrometry and NMR to be cyclic depsipeptide containing unusual amino acids, Ahp, and N-methyl tyrosine. As a result of protease inhibition test, streptopeptolin showed inhibitory activity against chymotrypsin. The whole genome sequence data of S. olivochromogenes revealed the biosynthetic gene cluster for streptopeptolin, which encoded a nonribosomal peptide synthetase. We proposed a biosynthetic pathway of streptopeptolin based on bioinformatics analysis.

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura.

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.

NMR analysis on the sialic acid-binding mechanism of an R-type lectin mutant by natural evolution-mimicry.

A sialic acid-binding lectin (SRC) was created from the C-terminal domain of an R-type N-acetyl lactosamine-binding lectin (EW29Ch) by natural evolution-mimicry. Here, we clarified its sialic acid-binding mechanism using NMR spectroscopy. The NMR analysis showed differences between conformations of the 6'-sialyllactose-bound SRC in the solution state and that in the crystal state, and differences between the internal motion of the loop region in subdomain γ in SRC and that of the corresponding region in EW29Ch. The NMR analysis thus provided useful information to explain the manner of binding to 6'-sialyllactose in solution, which the previous X-ray crystal structure analysis lacked.

Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand.

Using NMR spectroscopy and simulated annealing calculations, we determined the solution structure of the disulfide-linked cyclized decapeptide ACLPWSDGPC (SD), which is bound to an anti-(gibberellin A(4)) mAb 4-B8(8)/E9 and was found to be the first peptidyl mimotope for a hydrophobic ligand. The resulting structure of the peptide showed a beta-turn-like conformation in residues three to seven and the region converges well (average rmsd 0.54 A). The binding activity and the epitopes of the peptide to the antibody were assessed using saturation transfer difference (STD)-NMR experiments. We also conducted docking simulations between the peptide and the mAb to determine how the peptide is bound to the mAb. Resonances around the beta-turn-like conformation of peptide SD (residues 3-5) showed strong STD enhancement, which agreed well with results from docking simulation between peptide SD and the mAb. Together with the commonality of amino acid residues of the mAb involved in interactions with gibberellin A(4) (GA(4)) and peptide SD, we concluded that peptide SD is bound to the antigen-binding site of mAb 4-B8(8)/E9 as a GA(4) mimic, confirming evidence for the existence of peptide mimics even for hydrophobic ligands.