Abstract
Biosynthesis of the enediyne natural product calicheamicins γ1 I in Micromonospora echinospora ssp. calichensis is initiated by the iterative polyketide synthase (PKS) CalE8. Recent studies showed that CalE8 produces highly conjugated polyenes as potential biosynthetic intermediates and thus belongs to a family of highly-reducing (HR) type I iterative PKSs. We have determined the NMR structure of the ACP domain (meACP) of CalE8, which represents the first structure of a HR type I iterative PKS ACP domain. Featured by a distinct hydrophobic patch and a glutamate-residue rich acidic patch, meACP adopts a twisted three-helix bundle structure rather than the canonical four-helix bundle structure. The so-called ‘recognition helix’ (α2) of meACP is less negatively charged than the typical type II ACPs. Although loop-2 exhibits greater conformational mobility than other regions of the protein with a missing short helix that can be observed in most ACPs, two bulky non-polar residues (Met992, Phe996) from loop-2 packed against the hydrophobic protein core seem to restrict large movement of the loop and impede the opening of the hydrophobic pocket for sequestering the acyl chains. NMR studies of the hydroxybutyryl- and octanoyl-meACP confirm that meACP is unable to sequester the hydrophobic chains in a well-defined central cavity. Instead, meACP seems to interact with the octanoyl tail through a distinct hydrophobic patch without involving large conformational change of loop-2. NMR titration study of the interaction between meACP and the cognate thioesterase partner CalE7 further suggests that their interaction is likely through the binding of CalE7 to the meACP-tethered polyene moiety rather than direct specific protein-protein interaction.
Highlights
One of the most distinctive features of fatty acid, nonribosomal peptide and polyketide biosynthetic pathways is the utilization of the acyl carrier protein (ACP) or peptidyl carrier protein (PCP) for the shuttling of biosynthetic intermediates among various catalytic domains or proteins [1,2,3,4,5]
As the first structure determined for the ACP domain of a highly-reducing (HR) iterative type I polyketide synthase (PKS), meACP shares extremely low sequence homology with other known ACPs, including the ACP domain of the non-reducing (NR) iterative type I PKS norsolorinic acid synthase (NSAS) (12% identity). meACP features an unusual HMSSI motif (or H(L/M)(S/T/N)S(I/L) for meACP homologs) harboring the putative Ser971 phosphopantetheinylation site, in contrast to the more common GX(H/D)S(L/I) motif seen in other ACPs
The specificity towards Ser971 must be determined by the location of the residue and its proximity to the CoA substrate in the meACP-phosphopantetheinyl transferase (PPTase) complex
Summary
One of the most distinctive features of fatty acid, nonribosomal peptide and polyketide biosynthetic pathways is the utilization of the acyl carrier protein (ACP) or peptidyl carrier protein (PCP) for the shuttling of biosynthetic intermediates among various catalytic domains or proteins [1,2,3,4,5]. The small and highly dynamic ACPs or PCPs can be either free-standing proteins or integrated domains within a complex multidomain fatty acid synthase (FAS), polyketide synthase (PKS) or nonribosomal peptide synthase (NRPS). The ACP domains from the multidomain type I FASs adopt a similar overall structure, they do not seem to contain a pocket for binding and protecting the growing fatty acid chain [9,10,11]. Recent studies on the integrated ACP domain from the modular type I PKS DEBS and the discrete ACPs from type II PKS have revealed the similarity in overall protein structure but salient differences in the binding of acyl chain between type I and type II PKS ACPs [12,13,14,15,16,17]. Recent NMR studies further revealed functionally important protein dynamics in PCPs for the modulation of the interaction between the PCPs and the NRPS catalytic domains [5,18,19]
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