Type II polyketides include antibiotics such as tetracycline, and chemotherapeutics such as daunorubicin. Type II polyketides are biosynthesized by the type II polyketide synthase (PKS) that consists of 5 – 10 stand-alone domains. In many type II PKSs, the type II ketoreductase (KR) specifically reduce the C9-carbonyl group. How the type II KR achieves such a high regio-specificity, and the nature of stereo-specificity, are not well understood. Sequence alignment of KRs led to a hypothesis that a well-conserved 94-XGG-96 motif may be involved in controlling the stereochemistry. The stereo-specificity of single, double and triple mutant combinations of P94L, G95D and G96D were analyzed in vitro and in vivo for the actinorhodin KR (actKR). The P94L mutation is sufficient to change the stereospecificity of actKR. Binary and ternary crystal structures of both wild type and P94L actKR were solved. Together with assay results, docking simulations, and co-crystal structures, a model for stereochemical control is presented herein that elucidates how type II polyketides are introduced into the substrate pocket such that the C9-carbonyl can be reduced with high regio- and stereo-specificities. The molecular features of actKR important for regio- and stereo-specificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.
The
mechanistic details of many polyketide synthases (PKSs) remain
elusive due to the instability of transient intermediates that are
not accessible via conventional methods. Here we report an atom replacement
strategy that enables the rapid preparation of polyketone surrogates
by selective atom replacement, thereby providing key substrate mimetics
for detailed mechanistic evaluations. Polyketone mimetics are positioned
on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings
of substrate association upon nascent chain elongation and processivity.
Protein NMR is used to visualize substrate interaction with the actACP,
where a tetraketide substrate is shown not to bind within the protein,
while heptaketide and octaketide substrates show strong association
between helix II and IV. To examine the later cyclization stages,
we extended this strategy to prepare stabilized cyclic intermediates
and evaluate their binding by the actACP. Elongated monocyclic mimics
show much longer residence time within actACP than shortened analogs.
Taken together, these observations suggest ACP-substrate association
occurs both before and after ketoreductase action upon the fully elongated
polyketone, indicating a key role played by the ACP within PKS timing
and processivity. These atom replacement mimetics offer new tools
to study protein and substrate interactions and are applicable to
a wide variety of PKSs.
SUMMARY
Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs.
Novel chiral N-phosphinamide and N-phosphinyl imines have been designed, synthesized and applied to asymmetric aza-Henry reaction to give excellent chemical yields (92%- quant.) and diastereoselectivity (91% to >99%de). The reaction showed a great substrate scope in which aromatic/aliphatic aldehyde- and ketone-derived N-phosphinyl imines can be employed as electrophiles. The chiral N-phosphinamide can be stored at room temperature for more than 2 months without inert gas protection, and chiral N-phosphinyl imines were also proven to be highly stable at room temperature for a long period under inert gas protection. The N-phosphinyl group enabled the product purification to be performed simply by washing crude product with EtOAc and hexane. This reaction joined other eight GAP (Group-Assistant-Purification) chemistry processes that were developed in our laboratories. The absolute configuration has been unambiguously determined by converting a β-nitroamine product into a known N-Boc sample.
A variety of substituted chiral propargylamines have been synthesized by reacting chiral N-phosphonylimines with lithium aryl/alkyl acetylides. Seventeen examples were studied to give excellent yields (>90%) and diastereoselectivities (96 : 4 to 99 : 1). It was found that the types of bases for generating acetylides and solvents are crucial for effectiveness of this asymmetric reaction. In addition, N,N-isopropyl group on chiral N-phosphonylimine auxiliary was proven to be superior to other protecting groups in controlling diastereoselectivity.
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