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

Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.

To establish a method for assessing graft viability, in-vivo, following corneal transplantation.Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques.Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1%) and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7-35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage.In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo

Calculation of fluorescence contrast and signal to noise ratio of the calcein AM fluorescence staining.

<p>(A) 25x25 pixel arrays in viable (green square) and non-viable areas of the graft were used to calculate fluorescence contrast and signal to noise ratio. Scale bar 1mm. (B) The periphery of the graft was chosen as a standard area of non-viable tissue as this consistently contains areas of bare Descemet membrane as well as attached, non-viable cells; stained positively with ethidium homodimer (this images corresponds to the yellow square in Fig 2A). (C) Receiver-operator-characteristic for calcein AM fluorescence.</p

High magnification in-vivo, electron-microscopy and immuno-fluorescence live/dead imaging.

<p>(A) High magnification in-vivo images were taken using the Rostock corneal module. Individual cells and cell nuclei are clearly visible. Dead cells still attached to Descemet membrane (DM) (red arrow head) and bare areas of DM (green arrow can be seen), Scale bar 50μm. (B) The same patterns of cell loss (i.e. bare DM surrounded by dead cells) can be seen on trypan blue/alizarin red (Scale bar 50μm) viability staining and (C) scanning electron microscopy (100μm). (D) DMEK grafts triple stained with calcein AM blue, annexin V and ethidium show no overlap between calcein AM and early or late markers or apoptosis. Scale bar 100μm.</p

Experimental setup of DMEK surgical model used.

<p>(A) Image showing porcine eye mounted within holder and standard equipment used for DMEK surgery. (B) Tissue is imaged prior to and following DMEK preparation whilst still inside the standard Optisol viewing chamber. Use of the standard anterior segment lens supplied by the manufacturer and the 30° field-of-view imaging setting allows visualization of the entire cornea. (C) Image showing DMEK graft unfolded within the porcine anterior chamber. (D) The graft is imaged in-situ. The addition of microscope objective lens allows non-contact, individual cell imaging.</p

Optimization and characterization of calcein AM fluorescence for in-vivo imaging.

<p>(A) Calcein AM fluorescence shows a linear relationship with the incubation dose in cultured human corneal endothelial cells. (B) Peak fluorescence is seen at 2 hours after incubation. (C) Fluorscence dimishes rapidly over the first 24hrs in cells returned to culture. (D) Fluorescence dimishes rapidly over the first 24 hours (mean fluorescence drops from 109 to 36) in whole corneas stained with calcein AM and then returned to organ culture at 37°C, making descrimination between viable and non-viable areas not possible. For tissue stored in Optisol at 4°C, fluorescence contrast remains high at 7 days post incubation, with little change in fluorescence intensity (99 vs 89).</p