In vivo experiments involving the inactivation of proposed SgcE active sites were carried out, resulting in loss of C-1027 production by SgcE. Typically, PPTases are freestanding components in PKS biosynthetic pathways. A unique feature of this particular PKS is the presence of an integrated C-terminal PPTase that covalently attaches 4′-phosphopantetheine to the active site serine of the T domain. 124 Characterization of the biosynthetic gene cluster revealed the presence of a type I iterative PKS (SgcE) that catalyzes the formation of the nine-membered enediyene core from acyl-CoAs. The enediyne core is the key to the cytotoxicity of C-1027 because when it is released from the protein complex it can form a transient biradical species that can induce single-strand and double-strand breaks in DNA molecules. C-1027 is an extremely cytotoxic compound, isolated from Streptomyces globisporus, consisting of a binding protein (CagA) and a reactive nine-membered enediyne core containing a pair of conjugated acetylenic groups ( Figure 33(a)). MS has proven to be essential in investigations of the biosynthetic pathways of C-1027, an enediyne antitumor antibiotic.

20, 169 including 4,6-dehydration by SgcA ( 49 to 50), 3,5-epimerization by SgcA2 ( 50 to 139), C5-methylation by SgcA3 ( 139 to 140), C4-transamination by SgcA4 ( 140 to 141), N, N-dimethylation by SgcA5 ( 141 to 136), and coupling of 136 with the aglycone ( 135) by SgcA6. 169 Subsequent reactions were proposed to involve all other six enzymes as shown in Fig. 168,169 The initial step in the biosynthesis of the deoxy aminosugar of C-1027 is catalyzed by SgcA1 converting α- d-glucose-1-phosphate ( 48) and TTP to TDP-α- d-glucose ( 49).

169 Among the genes within the C-1027 biosynthetic gene cluster, there are seven deoxy aminosugar biosynthesis genes, sgcA (TDP-Glc-4,6-dehydratase), sgcA1 (TDP-glucose synthase), sgcA2 (TDP-4-keto-6-deoxyglucose epimerase), sgcA3 ( C-methyl transferase), sgcA4 (aminotransferase), sgcA5 ( N-methyltransferase), and sgcA6 (glycosyl transferase). Biosynthesis of the deoxy aminosugar moiety ( 136) of C-1027 was first studied by Shen and coworkers. The biosynthetic gene clusters for C-1027, 165 neocarzinostatin (NCS), 166 maduropeptin (MDP), 167 kedarcidin, 168 and calicheamicin, 69 have been cloned and sequenced, containing an iterative polyketide synthase (PKS) shared among these enediyne biosynthetic gene clusters. Proposed convergent assembly of C-1027 chromophore and the proposed biosynthesis of its deoxy aminosugar moiety. 65 Within the C-1027 BGC, 12 genes were predicted to be associated with the enediyne core, six with the β-tyrosine, seven with the benzoxazolinate, and seven with the 4-amino-6-deoxysugar biosynthesis, as well as one resistance gene, three regulatory genes and 20 genes of unknown function ( Fig. The sgcE gene, encoding the PKSE within the C-1027 BGC, was the first 9-membered enediyne PKSE to be identified, sequenced, and shown to be necessary for C-1027 production by gene inactivation experiments. 65 The C-1027 BGC spans ~ 85 kb DNA and consists of 67 genes, the sequencing of which revealed the unique enediyne PKS, PKSE, crucial for enediyne core biosynthesis. 64 Chromosomal walking from this locus led to the identification of the C-1027 BGC ( Fig. 54 This knowledge was utilized to identify a gene, sgcA encoding for a dNDP- d-hexose by a dNDP- d-glucose 4,6-dehydratase, clustered together with cagA, encoding the C-1027 apoprotein that had previously been cloned and sequenced. Identifying the C-1027 BGC involved searching for the genes encoding enzymes required to biosynthesize the 4-amino-6-deoxysugar in C-1027. The C-1027 BGC was cloned and sequenced in 2002.

Persamaan transistor c 1027