Biosynthetic Manipulation of Tryptophan in Bacteria: Pathways and Mechanisms

Tryptophan, the most chemically complex and the least abundant of the 20 common proteinogenic amino acids, is a biosynthetic precursor to a large number of complex microbial natural products. Many of these molecules are promising scaffolds for drug discovery and development. The chemical features of tryptophan, including its ability to undergo enzymatic modifications at almost every atom in its structure and its propensity to undergo spontaneous, non-enzyme catalyzed chemistry, make it a unique biological precursor for the generation of chemical complexity. Here, we review the pathways that enable incorporation of tryptophan into complex metabolites in bacteria, with a focus on recently discovered, unusual metabolic transformations

Desferrioxamine biosynthesis : diverse hydroxamate assembly by substrate-tolerant acyl transferase DesC

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in Streptomyces species involves the DesD-catalysed condensation of various N-acylated derivatives of N-hydroxycadaverine with two molecules of N-succinyl-N-hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N-acyl-N-hydroxycadaverine substrates of DesD from N-hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N-acylation of N-hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor. The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N-succinyl-N-hydroxycadaverine and one of N-acetyl-N-hydroxycadaverine) was also determined. This shows that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely functio

Antibiotic skeletal diversification via differential enoylreductase recruitment and module iteration in trans -acyltransferase polyketide synthases

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the “blind watchmaker” of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons

Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis

Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin

Structure, function and substrate tolerance of TxtC

Thaxtomins are a family of phytotoxins produced by Streptomyces scabies and other Streptomyces species, which cause scab disease in root crops, such as potato, carrot and radish. The final step of their biosynthesis involves sequential hydroxylation reactions catalysed by the cytochrome P450 (CYP), TxtC (figure 1). This CYP is of particular interest because it is chemically challenging to selectively hydroxylate at the phenylalanine alpha-position, and thaxtomins have been shown to have herbicidal activity. The hydroxyl groups introduced by TxtC have been shown to contribute both to the phytotoxicity, and solubility, of thaxtomin A. The first target was to complete a total chemoenzymatic synthesis of thaxtomin A. Peptide coupling of Boc-4-nitroTrp and N-Me-Phe methyl ester, cyclisation to give the monomethylated diketopiperazine and subsequent methylation of the 4-nitroTrp amide nitrogen led to thaxtomin D. TxtC was then utilised to complete the final two hydroxylation steps to give thaxtomin A as a single enantiomer. A range of TxtC substrate analogues were then synthesised in order to probe the SAR of TxtC. Reactivity was assessed both in vitro and in vivo and indicated a fairly broad substrate tolerance, with modification to the indole, phenyl group and N-methyl groups all tolerated to give monohydroxylated, and in some cases dihydroxylated, products. Selectivity was comparable to the natural substrate in those analogues with modification to the indole or N-methyl groups. A number of the hydroxylated products have been characterised by 1H and 13C NMR which show the major site of hydroxylation is as observed in thaxtomin A. X-ray crystallography of TxtC with both substrates, thaxtomin D and thaxtomin B, led to production of structures to 2.8Å and 1.7Å, respectively. These structures, as well as subsequent docking simulations, have given insights into the SAR observed, and the mechanism by which hydroxylation occurs

Binding of distinct substrate conformations enables hydroxylation of remote sties in thaxtomin D by cytochrome P450 TxtC

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation and natural product biosynthesis. Here we report biochemical, structural and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the -carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density cor-responding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-inter-mediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the -face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally-distinct binding mode in which the -hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the -hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohy-droxylated intermediate

Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis

The role played by Rieske non-heme iron-dependent oxygenases in natural product biosyntheses is reviewed, with particular focus on experimentally characterised examples.</p

Mechanistic insights into class B radical-S-adenosylmethionine methylases : ubiquitous tailoring enzymes in natural product biosynthesis

Class B radical S-adenosylmethionine (SAM) methylases are notable for their ability to catalyse methylation reactions in the biosynthesis of a wide variety of natural products, including polyketides, ribosomally biosynthesised and post-translationally modified peptides (RiPPs), nonribosomal peptides (NRPs), aminoglycosides, β-lactams, phosphonates, enediynes, aminocoumarins and terpenes. Here, we discuss the diversity of substrates and catalytic mechanism utilised by such enzymes, highlighting the stereochemical course of methylation reactions at un-activated carbon centres and the ability of some members of the family to catalyse multiple methylations

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480–500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results