The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.

The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology, and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the critical research discoveries relevant to the clinically most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clinical studies are also highlighted.

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The present study investigates whether compound I and compound II of manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium utilize the same Mn-binding site for catalysis. Manganese peroxidase was expressed from its cDNA in Escherichia coli and refolded from inclusion bodies to yield fully active enzyme. Three mutants of the enzyme were generated by site-directed mutagenesis. Each of the three amino acid residues proposed to be involved in Mn2+ binding, E35, D179, and E39, was mutated. The acidic side chains of E35 and E39 were shortened by one carbon to the acidic group D, and the acidic side chain of D179 was shortened by one carbon to the alkyl group A. These mutants, E35D, D179A, and E39D, were used to determine whether Mn2+ reacts at the same site with both compound I and compound II of manganese peroxidase and to determine whether phenolic substrates for the enzyme react at this site. Our results conclusively demonstrate that E35 and D179 residues are involved not only in Mn2+ binding but also in electron transfer from Mn2+ to the enzyme for both compound I and compound II. In contrast, E39 is not critically important to either process. None of the three residues is involved in reactions with phenolic substrates or with H2O2.

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A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3 untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.

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