Gaurav Shakya - Academia.edu (original) (raw)

Papers by Gaurav Shakya

ACS chemical biology, Jan 21, 2017

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with ... more Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a non-malonyl derived starter unit, which adds molecular diversity to the poly-β-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a sub-class ('DpsC type') of priming ketosynthases (KS III). To date, however, no structural information exists for this sub-class of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short...

Proceedings of the National Academy of Sciences of the United States of America, May 8, 2017

Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsi... more Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and ...

Angewandte Chemie International Edition, 2016

In fungal non-reducing polyketide synthases (NR-PKS), the acyl-carrier protein (ACP) carries the ... more In fungal non-reducing polyketide synthases (NR-PKS), the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanismbased crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-offunction ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides. Keywords Non-reducing polyketide synthase; crosslinking; acyl-carrier protein; product template domain Fungal polyketide natural products are chemically complex small molecules, many of which have diverse biological activities. Examples include the hepatocellular carcinogen aflatoxin

Journal of the American Chemical Society, Jan 3, 2014

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability... more 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 mu...

Chemistry & Biology, 2013

Protein,protein interactions, which often involve interactions among an acyl carrier protein (ACP... more Protein,protein interactions, which often involve interactions among an acyl carrier protein (ACP) and ACP partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal nonreducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ mechanism-based crosslinkers 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 mediates 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.

Chemical Biology & Drug Design, 2010

Novel chiral N-phosphinamide and N-phosphinyl imines have been designed, synthesized and applied ... more 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 b-nitroamine product into a known N-Boc sample.

Bioorganic & Medicinal Chemistry Letters, 2009

Chiral phosphonyl imines attached with N-isopropyl protection group were found to react with lith... more Chiral phosphonyl imines attached with N-isopropyl protection group were found to react with lithium glycine enolates under convenient conditions to give alpha,beta-diamino esters. Thirteen examples have been examined in good to excellent chemical yields (85-97%) diastereoselectivity (up to 99% de). By treating with HBr at room temperature, the chiral auxiliary can be readily removed and recycled. The absolute structure has been unambiguously determined by converting a product to a known sample.

Biochemistry, 2011

Type II polyketides include antibiotics such as tetracycline, and chemotherapeutics such as dauno... more 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 regiospecificity, 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 C9carbonyl 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. Streptomycete soil bacteria are one of nature's largest producers of clinically relevant pharmaceuticals (1, 2). A majority of these products belong to the polyketide family, which are used as antibiotic (actinorhodin), anti-cancer (daunorubicin), and immunosuppressive (FK506) agents (3). The wide range of bioactivities of polyketide natural products is due in part to the presence of multiple chiral centers that contribute to their structural diversity. Recently, the importance of developing chiral drugs and their effect on pharmacokinetics have been reviewed (4). Often, the specific bioactivity and biosynthesis of these natural products are dependent on the presence of chiral centers introduced in a controlled and systematic manner. In fact, many natural products lack biological activity in the absence of modifications such as hydroxylations and glycosylations that create chiral centers (5). The importance of chirality for the action and biosynthesis of polyketides therefore presents a new facet that can be controlled to develop new, therapeutic polyketide products. The chemical diversity of aromatic polyketides arises as a result of biosynthesis by polyketide synthase (PKS), which is closely related to fatty acid synthase (FAS). Both FAS