2.4.3 Enediyne Biosynthesis in the Genomic Era

As was revealed from the isotopic labeling experiments, little new insight was gained on the biosynthesis of the enediynes. Therefore, in the genomic era effort were undertaken to locate the putative type I or type II PKS gene on the genomic DNA of the enediyne producers (microorganisms) by DNA hybridization. However these were unsuccessful. Instead, Shen and coworkers succeeded in locating the DNA fragment that encompasses the enediyne gene clusters for C-1027. They were able to find out the gene (cagA) that encodes the chromophore-associated apoprotein CagA and two putatively conserved deoxysugar synthesizing genes. At the same time, Thorson et al. were able to identify the gene locus for calicheamicin biosynthesis. In 2002, the completion of the sequencing and partial annotation of the C-1027 and calicheamicin gene clusters was announced. Since then the study of enediyne biosynthesis entered the genomic era.

Therefore, genetic manipulations of genes related to secondary metabolism now offer a promising tool to investigate the biosynthetic pathway of formation of and prepare complex natural products like enediyne biosynthetically. This approach depends on the cloning and genetic and biochemical characterization of the biosynthetic pathways of the metabolites. Thus, several research groups are involved in the cloning, sequencing, and characterization of the several enediyne biosynthesis gene clusters from the producer microorganisms through which the convergent biosynthetic strategies for C-1027, NCS and other enediynes were developed. Manipulation of genes governing enediynes biosynthesis allowed one to engineer enediyne compounds. This approach offers the opportunity to decode the genetic and biochemical basis for the biosynthesis of enediynes and many other structurally complex natural products and to explore ways to make more antitumor agents. Here are few examples of biosynthesis of enediynes

2.4.3.1 The Apoprotein and The Gene Cluster for Enediyne Biosynthesis

The Apoprotein: All known 10-membered enediyne natural products were isolated as free-standing chromophores. On the contrary, the nine-membered enediynes were isolated as a chromoprotein complex- a binding protein known as protective apoprotein covering the dissociable enediyne chromophore. Exception is N1999A2 where no apoprotein was found. The 9-memred enediyne chromophores are extremely unstable in aqueous solution. For example neocarzinostatin is extremely unstable in aqueous solution in the absence of the apoprotein but the apoprotein cover greatly enhances the stability of the labile chromophore.

However the apoproteins are not similar for all the enediynes. As for example, the maduropeptin apoprotein (MdpA) does not share similarity with the apoproteins of neocarzinostatin, C-1027 and kedarcidin. The 133 amino acid long MdpA represents a new protein class that does not share significant sequence homology with any protein deposited in the National Center for Biotechnology Information (NCBI) database.

The structures of neocarzinostatin and C-1027 associated with their apoproteins (NcsA and CagA) have been determined which reveals that the apoproteins share an immunoglobulin-like fold that consists of a seven-stranded antiparallel β-barrel and two additional β-strands. The β-strands and the three loops of the apoprotein forms a hydrophobic pocket in which the chromophore (the reactive enediyne core) is accommodated. The structure of the protein–neocarzinostatin complex revealed that the reactive sites of the cromophore such as the epoxide, the acetylene groups, and the nucleophilic addition site are greatly shielded from the solvent. Similarly structure determination of the aromatized C-1027 chromophore revealed that the enediyne core and the the hydrophobic benzoxazolinate moiety interact and reside on the hydrophobic residues of the protein. These bindings bring stability of the extremely reactive 9-membered enediynes.

Role of Binding Proteins: The binding protein directs transport of the reactive enediyne chromophore to the extracellular environment. It is also found to be essential for self-resistance by stabilizing the reactive enediyne chromophore. The 9-membered enediynes C-1027 and maduropeptin readily undergo cycloaromatization in the absence of the binding protein. Thus the binding proteins help stabilizing the enediyne in their native form.

Establishment of the amino acid sequence of the homologous binding proteins enables the cloning and the sequencing of the genes-cagA for C-1027 and ncsA for NCS.

Since there is no binding protein for the ten-membered enediyne calicheamicin, a different strategy was used to clone the gene cluster of ten-membered enediyne calicheamicin. The strategy utilized to clone and localized the gene cluster of ten-membered enediyne calicheamicin  are-(a) screening of clones that are capable of conferring calicheamicin resistance using PCR-based screens and (b) followed up by DNA-shotgun sequencing.