Structural revision of sesbagrandiflorains A and B, and synthesis and biological evaluation of 6-methoxy-2-arylbenzofuran derivatives.

Sesbagrandiflorains A (1) and B (2), isolated from the stem bark of the Indonesian fabaceous plant Sesbania grandiflora, were reported to be 6-methoxy-2-(2´,3´-dihydroxy-5´-methoxyphenyl)-1-benzofuran-3-carbaldehyde and 6-hydroxy-2-(2´,3´-dihydroxy-5´-methoxyphenyl)-1-benzofuran-3-carbaldehyde, respectively. However, based on reevaluation of their 1D and 2D NMR data, the chemical structures of 1 and 2 have been revised to 4-hydroxy-2-(4´-hydroxy-2´-methoxyphenyl)-6-methoxybenzofuran-3-carbaldehyde and 4-hydroxy-2-(4´-hydroxy-2´-hydroxyphenyl)-6-methoxybenzofuran-3-carbaldehyde, respectively. In addition, seven new derivatives of 1 have been synthesized from the natural product in good yields (65 - 93%). The chemical structures of the synthetic compounds-one diester (6), four ethers (7-10), one secondary amine (11), and one oxime (12)-were confirmed by MS and NMR analysis. Compound 6 exhibited moderate antibacterial activity against the plant pathogen Rhodococcus fascians with a MIC of 0.1 mg/mL. Compounds 8 and 12 demonstrated respectable cytotoxicity against A375 melanoma cancer cells line with the relative IC50 values of 22.8 and 32.7 µM, respectively.

Biosynthesis of the Nuclear Factor of Activated T Cells Inhibitor NFAT-133 in Streptomyces pactum.

NFAT-133 is a Streptomyces-derived aromatic polyketide compound with immunosuppressive, antidiabetic, and antitrypanosomal activities. It inhibits transcription mediated by nuclear factor of activated T cells (NFAT), leading to the suppression of interleukin-2 expression and T cell proliferation. It also activates the AMPK pathway in L6 myotubes and increases glucose uptake. In addition to NFAT-133, a number of its congeners, e.g., panowamycins and benwamycins, have been identified. However, little is known about their modes of formation in the producing organisms. Through genome sequencing of Streptomyces pactum ATCC 27456, gene inactivation, and genetic complementation experiments, the biosynthetic gene cluster of NFAT-133 and its congeners has been identified. The cluster contains a highly disordered genetic organization of type I modular polyketide synthase genes with several genes that are necessary for the formation of the aromatic core unit and tailoring processes. In addition, a number of new analogs of NFAT-133 were isolated and their chemical structures elucidated. It is suggested that the heptaketide NFAT-133 is derived from an octaketide intermediate, TM-123. The current study shows yet another unusual biosynthetic pathway involving a noncanonical polyketide synthase assembly line to produce a group of small molecules with valuable bioactivities.

Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis.

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free ß-ketoacid that subsequently undergoes decarboxylation.

Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

Pactamycin, a structurally unique aminocyclitol natural product isolated from Streptomyces pactum, has potent antibacterial, antitumor, and anti-protozoa activities. However, its production yields under currently used culture conditions are generally low. To understand how pactamycin biosynthesis is regulated and explore the possibility of improving pactamycin production in S. pactum, we investigated the transcription regulations of pactamycin biosynthesis. In vivo inactivation of two putative pathway-specific regulatory genes, ptmE and ptmF, resulted in mutant strains that are not able to produce pactamycin. Genetic complementation using a cassette containing ptmE and ptmF integrated into the S. pactum chromosome rescued the production of pactamycin. Transcriptional analysis of the ΔptmE and ΔptmF strains suggests that both genes control the expression of the whole pactamycin biosynthetic gene cluster. However, attempts to overexpress these regulatory genes by introducing a second copy of the genes in S. pactum did not improve the production yield of pactamycin. We discovered that pactamycin biosynthesis is sensitive to phosphate regulation. Concentration of inorganic phosphate higher than 2 mM abolished both the transcription of the biosynthetic genes and the production of the antibiotic. Draft genome sequencing of S. pactum and bioinformatics studies revealed the existence of global regulatory genes, e.g., genes that encode a two-component PhoR-PhoP system, which are commonly involved in secondary metabolism. Inactivation of phoP did not show any significant effect to pactamycin production. However, in the phoP::aac(3)IV mutant, pactamycin biosynthesis is not affected by external inorganic phosphate concentration.

A Highly Promiscuous ß-Ketoacyl-ACP Synthase (KAS) III-like Protein Is Involved in Pactamycin Biosynthesis.

ß-Ketoacyl-acyl carrier protein (ß-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein (PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products.

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.