Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit

Background: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therape utically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productiv e interaction between these two parallel enzymatic systems has recently bee n demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, ep othilone D from great potential as an anti-tumor Sorangium cellulosum, has shown great potential as an anti-tumor agent. Results: The chain-initiating methylthiazole ring of epothilone has been ge nerated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier prot ein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four- domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carri er protein domains: CyA-Ox-PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH intro duced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loa ding with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate. Wh en acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of Ep oB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB , generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazol inyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy -S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-ba sed priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB a cyl enzymes were produced. Conclusions: These experiments establish chain transfer across a PKS and NR PS interface, Transfer of the acetyl group from the ACP domain of EpoA to E poB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocyc le during this natural product chain growth. (C) 2001 Elsevier Science Ltd. All rights reserved.