Satoshi Takamatsu

Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone–type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis—the producer of the important anthelmintic agent avermectin with annual world sales of

Genome mining in Streptomyces avermitilis : a biochemical Baeyer-Villiger reaction and discovery of a new branch of the pentalenolactone family tree

850 million—and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.

Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi-isozizaene metabolite.

Incubation of 1-deoxy-11-oxopentalenic acid (12) with recombinant PtlE protein from Streptomyces avermitilis in the presence of NADPH and catalytic FAD gave the Baeyer-Villiger oxidation product, the previously unknown compound neopentalenolactone D (13), representing a new branch of the pentalenolactone biosynthetic pathway. The structure and stereochemistry of the derived neopentalenolactone D methyl ester (13-Me) were fully assigned by a combination of GC-MS and NMR analysis and confirmed by X-ray crystallography. Neopentalenolactone D (13) was also isolated from engineered cultures of S. avermitilis from which the ptlD gene within the 13.4-kb (neo)-ptl biosynthetic gene cluster had been deleted. The DeltaptlEDeltaptlD double deletion mutant accumulated 12, the substrate for the ptlE gene product, while the corresponding single DeltaptlE mutant produced 12 as well as the related oxidation products 14 and 15. Engineered strains of S. avermitilis, SUKA5 and pKU462::ermRp-ptl cluster, harboring the complete (neo)ptl cluster produced the oxidized lactone 18 and the closely related seco acid hydrolysis products 16 and 17.

Regio- and stereospecificity of filipin hydroxylation sites revealed by crystal structures of cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The genome‐sequenced, Gram‐positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi‐isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi‐isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg2+‐dependent cyclization of farnesyl diphosphate to epi‐isozizaene. To effect the production of epi‐isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large‐deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi‐isozizaene. The previously characterized oxidized epi‐isozizaene metabolites (4R)‐ and (4S)‐albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi‐isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450‐encoding sav3031. This new metabolite was identified as 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol which is most likely formed by oxidation of (4S)‐albaflavenol.

Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.

Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis

Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462∷ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462∷ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.

Relative and absolute stereochemistry of the melanogenesis inhibitors OH-3984 K1 and K2. Partial synthesis from albocycline

The first total synthesis of extracellular factor, “Avenolide”, in Streptomyces avermitilis has been achieved using a convergent approach. The stereogenic centers in two key segments were installed using Sharpless epoxidation and dihydroxylation. This synthetic study allowed the determination of the absolute configuration of avenolide as 4S,10R, and yielded important information on its structure–activity relationship.

Screening for Free Radical Scavenging and Cell Aggregation Inhibitory Activities by Secondary Metabolites from Turkish Verbascum species

The transformation of the macrolide antibiotic albocycline (3) to the recently isolated melanin synthesis inhibitors OH-3984 K1 (1 and K2 (2) has established the relative and absolute configurations of 1 (4R, 7R, 12S) and 2 (4R, 7R, 12S, 13R).

Introduction of a benzoyl group onto riboside in aqueous solution: One-step synthesis of 6-chloropurine 2′,3′-di-O-benzoylriboside

Free radical scavenging and cell aggregation inhibitory activities of 36 secondary metabolites isolated from the methanolic extracts of Verbascum cilicicum Boiss., V. lasianthum Boiss. ex Bentham, V. pterocalycinum var. mutense Hub.-Mor., and V. salviifolium Boiss. (Scrophulariaceae) were investigated. The isolated compounds, 6-O-vaniloyl ajugol (1), ilwensisaponin A (2), ilwensisaponin C (3), verbascoside (4), β-hydroxyacteoside (5), martynoside (6), poliumoside (7), forsythoside B (8), angoroside A (9), dehydrodiconiferyl alcohol-9-O-β-D-glucopyranoside (10), dehydrodiconiferyl alcohol-9′-O-β-D-glucopyranoside (11), apigenin 7-O-β- glucopyranoside (12), luteolin 7-O-β-glucopyranoside (13), luteolin 3′-O-β-glucopyranoside (14) and chrysoeriol 7-O-β-glucopyranoside (15), exhibited a dose-dependent inhibition of bioautographic and spectrophotometric DPPH activities. Verbascoside (4) was the most active (IC50 4.0 μg/ml) comparing it to vitamin C (IC50 4.4 μg/ml) to inhibit phorbol 12-myristate 13-acetate (PMA)-induced peroxide-catalyzed oxidation of 2′,7′-dichlorofluorescein (DCFH) by reactive oxygen species (ROS) within human promyelocytic HL-60 cells. Ilwensisaponin A (2) (MIC 6.9 μg/ml) showed moderate in vitro activity on lymphocyte-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1)-mediated aggregation using the HL-60 cell line [positive control was cytochalasin B (MIC 2.3 μg/ml)]. None of the other compounds showed free radical scavenging and cell aggregation inhibitory activities.

Corrigendum: Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis

Abstract A benzoyl group was introduced onto the 3′-hydroxyl group of 6-chloropurine riboside by treatment with benzoylating agents in the presence of an organic or inorganic base in aqueous solution, in which further reaction gave 6-chloropurine 2′,3′-di- O -benzoylriboside.