Nonribosomal pathway

Heterocycles are found in a wide variety of natural products, and the chemical nature of these moieties imparts recognition elements critical to both protein and nucleic acid targets. These moieties may be biosynthesized via either ribosomal or nonribosomal pathways and can occur either singly within a molecule or as multiple, repeating heterocyclic units within the same compound (Figure 13.19). 

In bacterial cells and in yeast, which are, for obvious reasons, much more commonly studied than other animals and plants, amino acids, synthesized de novo or by degradation of proteins of various kinds and sizes, are found in the cytosol. The amino acids from these proteins are used for the synthesis of peptides. Much of the synthesis occurs either on/in the ribosomes of the endoplasmic reticulum (ER) or through nonribosomal pathways using acyl carrier protein-like condensation reactions. 

The nonribosomal pathway may be operational alongside the ribosomal pathway outlined above, but it is independent of mRNA and, rather than producing proteins (peptides) obviously required for life, a different set of peptides (called secondary... 

Scheme 12.73. A flow diagram-type scheme showing the overall use of the modules present and required to effect peptide synthesis utilizing a nonribosomal pathway.

During the biosynthesis of nonribosomal peptides, there are two ways to incorporate the nonprotein amino acids. They can be incorporated either as a single unit or as an L-a-amino acid, which then undergoes structural modifications, while attached to the carrier protein. In the case of coronamic acid, L-rr//o-isoleucine is loaded onto the carrier protein and a unique biosynthetic pathway produces a cyclopropyl group containing a nonprotein amino acid. Specific examples of the biosynthesis of nonprotein amino acids will be discussed in the following sections. 

A number of nonprotein amino acids with unsaturated side chains have been isolated. Many of these contain alkene side chains, but some alkyne side chains containing amino acids have also been identified. Nonprotein dehydroamino acids do not have an a-stereocenter these amino acids are still classified under this category. Dehydroamino acids are generally biosynthesized by the enzymatic elimination of a leaving group at the /3-carbon. For example, serine and threonine are enzymatically dehydrated to give dehydroalanine and dehydrobutyrine, respectively. A similar biosynthetic pathway is hypothesized for dehydroamino acids found in nonribosomal peptides, such as nodularins and microcystins. ... 

The glycopeptide antibiotics such as vancomycin and chloroeremomycin are complex nonribosomal peptides. One of the nonprotein amino acids found in these antibiotics is 4-hydroxyphenylglycine. The biosynthetic pathway of this nonprotein amino acid has been studied and prephenate was... 

Based on our current understanding of ribosomal protein synthesis, several strategies have been developed to incorporate amino acids other than the 20 standard proteinogenic amino acids into a peptide using the ribosomal machinery . This allows for the design of peptides with novel properties. On the one hand, such a system can be used to synthesize nonstandard peptides that are important pharmaceuticals. In nature, such peptides are produced by nonribosomal peptide synthetases, which operate in complex pathways. On the other hand, non-natural residues are a useful tool in biochemistry and biophysics to study proteins. For example, incorporation of non-natural residues by the ribosome allows for site-specific labeling of proteins with spin labels for electron paramagnetic resonance spectroscopy, with... 

Chakraborty RN, Patel HN, Desai SB (1990) Isolation and Partial Characterization of Catechol-Type Siderophore from Pseudomonas stutzeri. Curr Microbiol 20 283 Challis GL (2005) A Widely Distributed Bacterial Pathway for Siderophore Biosynthesis Independent of Nonribosomal Peptide Synthetases. ChemBioChem 6 601 Chambers CE, McIntyre DD, Mouck M, Sokol PA (1996) Physical and Structural Characterization of Yersiniophore, a Siderophore Produced by Clinical Isolates of Yersinia entero-colitica. BioMetals 9 157... 

A final example of metabolic pathway engineering is based on polyketide and nonribosomal peptide biosynthesis. Polyketides and nonribosomal peptides are complex natural products with numerous chiral centers, which are of substantial economic benefit as pharmaceuticals. These natural products function as antibiotics [erythromycin A (65), vancomycin (66)], antifungals (rapamycin, amphotericin B), antiparasitics [avermectin Ala (67)], antitumor agents [epothiolone A (68), calicheamicin yj, and immunosuppressants [FK506 (69), cyclosporin A], Because this exponentially growing and intensely researched field has developed, the reader is directed to review articles for additional details.347-359 Also with the potential economic benefit to develop the next blockbuster pharmaceutical, a number of patents and patent applications have been published.360-366... 

Because these genes are often arranged in clusters, this has allowed a combinatorial biosynthetic approach for the construction of diverse libraries of polyketides and nonribosomal peptides.388387 The use of E. coli as a production host whereby precursor supply and selected pathway enzymes are modified has resulted in respectable titers of targeted compounds as well as novel derivatives.388393... 

The polyketides (23) form the only major class of natural products that has monomer couplings that do not involve the loss of phosphate like the nonribosomal peptides, the monomer couplings use a thioester intermediate. These molecules, which have biosynthetic pathways much like those of fatty acids, are assembled from the alpha-carboxylated two- and three-carbon metabolites malonyl-CoA and methylmalonyl-CoA (22). In these monomers, the alpha-carboxyl group is a caged form of carbon dioxide, and decarboxylation yields a thioester enolate... 

Figure 3 Biosynthetic pathways. (A) In the terpenoid coupling reaction, isomers of isopentenyl pyrophosphate are joined with the loss of pyrophosphate, leading to a linear intermediate that is cyclized to a terpenoid skeleton, as shown for the diterpene taxol. (B) In the polysaccharide coupling reaction, hexose and pentose monomers are joined with the loss of a nucleoside diphosphate, as shown for the epivancosaminyl-glucose disaccharide of vancomycin. (C) In the first step of the nonribosomal peptide coupling reaction, an aminoacyl adenylate is transferred to a carrier protein or thiolation domain (denoted T ) with loss of adenosine monophosphate. In the second step, this carrier protein-tethered aminoacyl group is coupled to the amine of an aminoacyl cosubstrate, forming a peptide bond, as shown for two residues in backbone of vancomycin. (D) In the polyketide coupling reaction, the loss of carbon dioxide from a two or three-carbon monomer yields a thioester enolate that attacks a carrier protein-tethered intermediate, forming a carbon-carbon bond as shown for the polyketone precursor of enterocin.

The mammalian multifunctional fatty acid synthase is a member of a large family of complex enzymes termed megasynthases that participate in step-by-step synthetic pathways. Two important classes of compounds that are synthesized by such enzymes are the polyketides and the nonribosomal peptides. The antibiotic erythromycin is an example of a polyketide, whereas penicillin (Section 8.5.5) is a nonribosomal peptide. 

Equally important as NMR spectroscopy is the development of mass spectrometry where advances have been even more spectacular, partly due to its far greater sensitivity. A broad introduction is provided by Hocart (Chapter 9.10), who describes the range of available spectrometers and techniques. In the next chapter (Chapter 9.11), Dorrestein and his coauthors describe applications to the elucidation of biosynthetic pathways, focusing on nonribosomal peptides and polyketides, while Das provides a detailed account of the structure determination of proteins and peptides in Chapter 9.12. In yet another illustration of the power of mass spectrometry to analyze biomacromolecules, Li and Altman extend the technique to a study of bacterial polysaccharides (Chapter 9.13). 

The emphasis of this review is placed on two structural classes of natural products polyketides and nonribosomal peptides (NRPs). The MS of these biosynthetic pathways is most advanced and will be covered in detail. In the following sections we will describe the current methods and applications used to study the biosynthetic pathways of natural products and provide a glimpse into upcoming techniques. In addition, a brief introduction to experimental design using high-end MS to study the biosynthesis of other natural metabolites, such as ribosomally encoded pathways and cofactors, is described. 

CPs are integral components of various primary and secondary metabolic pathways, including fatty acid synthesis (FAS), nonribosomal peptide synthesis (NRPS), polyketide synthesis (PKS), and lysine biosynthesis. All CPs harbor... 

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