Solution Structures of the Acyl Carrier Protein Domain from the Highly Reducing Type I Iterative Polyketide Synthase CalE8

Biosynthesis of the enediyne natural product calicheamicins γ1I 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

A study of the functional domains of the type I iterative polyketide synthase CalE8 in calicheamicin biosynthesis.

Naturally occurring enediynes are potent antibiotics produced by soil and marine microorganisms. Their robust antitumor activities and unique mode of action make them a significant topic of study. The synthesis of the enediyne products is initiated by a type I iterative polyketide synthase (PKS). In this project, we examine the structures and functions of the domains of CalE8, to demarcate the domains of the the iterative PKS from the biosynthetic pathway of the 10-membered enediyne calicheamicin in Micromonospora echinospora spp.DOCTOR OF PHILOSOPHY (SBS

A study of the functional domains of the type I iterative polyketide synthase CalE8 in calicheamicin biosynthesis

Naturally occurring enediynes are potent antibiotics produced by soil and marine microorganisms. Their robust antitumor activities and unique mode of action make them a significant topic of study. The synthesis of the enediyne products is initiated by a type I iterative polyketide synthase (PKS). In this project, we examine the structures and functions of the domains of CalE8, to demarcate the domains of the the iterative PKS from the biosynthetic pathway of the 10-membered enediyne calicheamicin in Micromonospora echinospora spp. The 212 KDa CalE8 contains several domains including the predicted ketoacyl synthase (KS), acyl transferase (AT), ketoreductase (KR) and dehydratase (DH) domains. In addition, CalE8 also contains a postulated acyl carrier protein (ACP) domain and a C-terminal domain with unknown function. The first step of enediyne biosynthesis involves a post-translational modification of the ACP domain by 4’- phosphopantetheinylation. The ACP domain and C-terminal portion of CalE8 were first cloned and expressed as stand-alone proteins to study their functions. The identity of the ACP domain was established by in vitro phosphopantetheinylation using the surfactin PPTase (Sfp) from Bacillus subtilis. The NMR solution of the ACP domain was solved to show that the ACP exhibits some rather distinct structure feature from other ACPs. Furthermore, we found that the C-terminal domain exhibits PPTase activity towards various carrier proteins. Sequence analysis and modeling studies suggest the C-terminal domain is an unusual Sfp-like PPTase domain integrated into CalE8. Finally, the AT, KS, DH and KR domains of the CalE8 PKS were examined thoroughly by bioinformatics tools such as structural modeling to define the domain boundaries and catalytic residues. The individual domains were cloned and expressed in E. coli for structure determination. Although the KS, DH and KR domain proteins were found to be insoluble, the AT domain was soluble and purified for further studies. The access to the soluble AT domain will be valuable for studying the substrate specificity of the AT domain in accepting malonyl-CoA, but not acetyl-CoA as substrate. For characterizing the covalently-attached products of CalE8, an ACP phosphodiesterase capable of cleaving the growing polyketides was examined. This family of phosphodiesterases was found to be highly unstable with the propensity to form precipitate in solution. We have identified a phosphodiesterase (PaAcpH) from Pseudomonas aeruginosa that can be expressed and purified as a soluble protein. The function and substrate specificity of PaAcpH was first validated and examined with several carrier proteins from different pathways. We demonstrate that PaAcpH is indeed able to catalyze the removal of the phosphopantetheinyl moiety and the tethered-intermediates from CalE8. The capability of releasing the polyketide intermediates by PaAcpH is valuable in the study of PKS mechanisms.DOCTOR OF PHILOSOPHY (SBS

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

AbstractThe polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp

AbstractProtein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer’s, Parkinson’s and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitroTGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611–633 with the mutation c.623 G&gt;R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611–633 c.623 G&gt;R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.</jats:p