Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway, and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3–1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.
β-Ketoacyl-acyl carrier protein (β-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.
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.
Glycosylation is a common modification reaction in natural products 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 3aminoacetophenone 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 β-ketoacid that subsequently undergoes decarboxylation.Glycosylation is one of the most ubiquitous and important transformations in nature and plays a central role in the structural and physiological aspects of living organisms. Glycosyltransferases are the family of enzymes that catalyze glycosylation, resulting in sugar-containing products. In natural products biosynthesis, glycosylation is generally considered to be a tailoring process that takes place later in the pathway after the backbone structure is formed. Some exceptions apply to certain natural products when glycosylation is directly involved in the formation of the core structure. Such phenomenon has been proposed to occur in the biosynthesis of at least two highly important microbial natural products, mitomycin and pactamycin. [1][2][3]
Pactamycin is a structurally unique aminocyclitol antibiotic with broad-spectrum cell growth inhibitory activity. To explore the bountiful activity of the aminocyclitol core of pactamycin, an efficient, modular, and asymmetric synthesis of aminocyclopentitols resembling the pactamycin pharmacophore has been developed employing a SmI-mediated imino-pinacol coupling strategy. Two of the compounds exhibited antitumor activity against A375 melanoma cells.
The cover picture shows the complex biosynthetic pathway to the antitumor antibiotic pactamycin in the soil bacterium Streptomyces pactum. Previous studies have suggested that some of the tailoring enzymes in pactamycin biosynthesis have relaxed substrate specificity that can lead to unspecific modifications of the intermediates. The activity of these promiscuous enzymes not only makes it difficult to determine the actual sequence of the pathway, but also impedes the production of structurally less complex analogues of pactamycin. In this study, a combination of genetic engineering and metabolic analysis was used to gain insights into the natural sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. Two new analogues that have better selectivity indices than pactamycin against malarial parasites were also identified. Artist: Seika Mahmud. More information can be found in the communication by T. Mahmud et al. on page 1585 in Issue 17, 2016 (DOI: 10.1002/cbic.201600261).
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