The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here we report a comprehensive set of genetic and enzymatic experiments that establish functions for the seventeen moenomycin biosynthetic genes involved in the synthesis moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogs. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogs can be explored.
Many secondary metabolites of clinical importance have been isolated from different Streptomyces species. As most of the natural producers remain difficult to handle genetically, heterologous expression of an entire biosynthetic gene cluster in a well characterised host allows improved possibilities for modifications of the desired compound by manipulation of the biosynthetic genes. However, the large size of a functional gene cluster often prevents its direct cloning into a single cosmid clone. Here we describe a successful strategy to assemble the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) into a single cosmid clone by lambda RED recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO--responsible for the C-methylation at the 8-positions of the aminocoumarin moieties in coumermycin A1--and heterologous expression of the modified cluster resulted in an accumulation of a C-8-unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two chlorine atoms. The identities of the new compounds were verified by LC-MS, and their antibacterial activities were tested against Bacillus subtilis in an agar diffusion assay.
The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.Nature uses prenyltransferases (PTs) to make a wide variety of primary and secondary metabolites containing prenyl groups, and there is enormous interest in understanding the scope and utility of these enzymes. 1 The best studied classes of PTs, the polyprenyl synthases and the cyclizing terpene synthases, contain an alpha helical fold, as do the protein farnesyl and geranylgeranyl transferases. 2 Recently, the first PT having a triosephosphate isomerase (TIM) barrel fold was identified in archaea. [3][4][5] This PT, geranylgeranylglyceryl phosphate synthase (GGGPS), catalyzes the transfer of a geranylgeranyl (C20) group to glycerol-1-phosphate (G1P) during the first step in the biosynthesis of the major phospholipid component of archaeal membranes (Figure 1). 6,7 We recently discovered a bacterial GGGPS homolog, MoeO5, in the gene cluster for the antibiotic moenomycin. MoeO5 couples 3-phosphoglyceric acid (3PG) to a farnesyl group to form the starter unit for moenomycin biosynthesis (Figure 1). 8,9 Here, we study the mechanism of MoeO5, describe key residues that can be used to predict the acceptor substrate in TIM barrel PTs, apply these criteria to a bacterial GGGPS homolog of unknown function, PcrB, and establish its substrates biochemically. We conclude that TIM barrel PTs are dedicated to the transfer of prenyl groups to oxygen on phosphorylated triose acceptors, but the reactions they catalyze can proceed by two very different mechanisms. The TIM barrel PTs, GGGPS and MoeO5, both make ether-linked products but with different allylic bond geometries (Figure 1). Unlike the GGGPS product, which retains the trans-geometry of the starting material and likely proceeds by a straightforward displacement mechanism, the MoeO5 product (6) contains a cis-allylic double bond. 8 MoeO5 is the only known prenyltransferase that forms an intermolecular cis-allylic bond using a trans-allylic precursor. 10 This linkage suggests that bond isomerization occurs prior to coupling during the catalytic reaction, and a possible reaction mechanism...
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