Ashish Garg

Bio: Ashish Garg is an academic researcher from Brown University. The author has contributed to research in topics: Enantioselective synthesis & Lewis acids and bases. The author has an hindex of 9, co-authored 11 publications receiving 313 citations. Previous affiliations of Ashish Garg include Indian Institute of Technology Kanpur & University of Texas at Austin.

Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase

Abstract: The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixtu...

Coupled methyl group epimerization and reduction by polyketide synthase ketoreductase domains. Ketoreductase-catalyzed equilibrium isotope exchange.

Abstract: Incubation of [2-(2)H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP(+) (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-(2)H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-(2)H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.

Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange.

Abstract: Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.

Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues

Abstract: The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-2) and 0.05 equiv of NADP+ in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography–tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substr...

Methods in Enzymology.

Abstract: I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Recent developments in solvent-free multicomponent reactions: a perfect synergy for eco-compatible organic synthesis

Abstract: Multicomponent reactions have gained significant importance as a tool for the synthesis of a wide variety of useful compounds, including pharmaceuticals. In this context, the multiple component approach is especially appealing in view of the fact that products are formed in a single step, and the diversity can be readily achieved simply by varying the reacting components. The eco-friendly, solvent-free multicomponent approach opens up numerous possibilities for conducting rapid organic synthesis and functional group transformations more efficiently. Additionally, there are distinct advantages of these solvent-free protocols since they provide reduction or elimination of solvents thereby preventing pollution in organic synthesis “at source”. The chemo-, regio- or stereoselective synthesis of high-value chemical entities and parallel synthesis to generate a library of small molecules will add to the growth of multicomponent solvent-free reactions in the near future. In this review we summarized the results reported mainly within the last 10 years. It is quite clear from the growing number of emerging publications in this field that the possibility to utilize multicomponent technology allows reaction conditions to be accessed that are very valuable for organic synthesis. Therefore, diversity oriented synthesis (DOS) is rapidly becoming one of the paradigms in the process of modern drug discovery. This has spurred research in those fields of chemical investigation that lead to the rapid assembly of not only molecular diversity, but also molecular complexity. As a consequence multi-component as well as domino or related reactions are witnessing a new spring.

On the Structure of "秀才秀才,错字布袋

Abstract: Sentence patterns like "秀才秀才,错字布袋"are unique in the grammatical structure, semantic structure and pragmatic function. The typical feature of this pattern is that the same word or phrase reappears continually at the very beginning. It has two parts: (1) The proceeding part("秀才秀才") includes a word and its repeated form, which is different from the reduplication in grammar and the continual repetition in rhetoric. This part can have referential functions in particular situations;and (2) The main function of the last part ("错字布袋")is to interpret the proceeding one. It is the semantic focus of the whole sentence. This sentence pattern typically shows the features of proverbs like "秀才秀才,错字布袋"in language structure,semantic meaning and pragmatic function.