Nair SK

References (6)

Title : Structural Basis for Enzymatic Off-Loading of Hybrid Polyketides by Dieckmann Condensation - Cogan_2020_ACS.Chem.Biol_15_2783
Author(s) : Cogan DP , Ly J , Nair SK
Ref : ACS Chemical Biology , 15 :2783 , 2020
Abstract : While several bioactive natural products that contain tetramate or pyridone heterocycles have been described, information on the enzymology underpinning these functionalities has been limited. Here we biochemically characterize an off-loading Dieckmann cyclase, NcmC, that installs the tetramate headgroup in nocamycin, a hybrid polyketide/nonribosomal peptide natural product. Crystal structures of the enzyme (1.6 A) and its covalent complex with the epoxide cerulenin (1.6 A) guide additional structure-based mutagenesis and product-profile analyses. Our results offer mechanistic insights into how the conserved thioesterase-like scaffold has been adapted to perform a new chemical reaction, namely, heterocyclization. Additional bioinformatics combined with docking and modeling identifies likely candidates for heterocycle formation in underexplored gene clusters and uncovers a modular basis of substrate recognition by the two subdomains of these Dieckmann cyclases.
ESTHER : Cogan_2020_ACS.Chem.Biol_15_2783
PubMedSearch : Cogan_2020_ACS.Chem.Biol_15_2783
PubMedID: 33017142
Gene_locus related to this paper: 9pseu-NcmC

Title : Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants - Chekan_2017_Proc.Natl.Acad.Sci.U.S.A_114_6551
Author(s) : Chekan JR , Estrada P , Covello PS , Nair SK
Ref : Proc Natl Acad Sci U S A , 114 :6551 , 2017
Abstract : Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.
ESTHER : Chekan_2017_Proc.Natl.Acad.Sci.U.S.A_114_6551
PubMedSearch : Chekan_2017_Proc.Natl.Acad.Sci.U.S.A_114_6551
PubMedID: 28584123
Gene_locus related to this paper: 9cary-r4p353

Title : Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates - Chekan_2016_J.Am.Chem.Soc_138_16452
Author(s) : Chekan JR , Koos JD , Zong C , Maksimov MO , Link AJ , Nair SK
Ref : Journal of the American Chemical Society , 138 :16452 , 2016
Abstract : Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs), with a threaded knot structure that is formed by an isopeptide bond attaching the N-terminus of the peptide to a side chain carboxylate. Some lasso peptide biosynthetic clusters harbor an enzyme that specifically hydrolyzes the isopeptide bond to yield the linear peptide. We describe here the 2.4 A resolution structure of a lasso peptide isopeptidase revealing a topologically novel didomain architecture consisting of an open beta-propeller appended to an alpha/beta hydrolase domain. The 2.2 A resolution cocrystal structure of an inactive variant in complex with a lasso peptide reveals deformation of the substrate, and reorganization of the enzyme active site, which exposes and orients the isopeptide bond for hydrolysis. Structure-based mutational analysis reveals how this enzyme recognizes the lasso peptide substrate by shape complementarity rather than through sequence specificity. The isopeptidase gene can be used to facilitate genome mining, as a network-based mining strategy queried with this sequence identified 87 putative lasso peptide biosynthetic clusters, 65 of which have not been previously described. Lastly, we validate this mining approach by heterologous expression of two clusters encoded within the genome of Asticcaucalis benevestitus, and demonstrate that both clusters produce lasso peptides.
ESTHER : Chekan_2016_J.Am.Chem.Soc_138_16452
PubMedSearch : Chekan_2016_J.Am.Chem.Soc_138_16452
PubMedID: 27998080
Gene_locus related to this paper: astec-e8rup5

Title : Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes - Zhang_2014_Proc.Natl.Acad.Sci.U.S.A_111_E3708
Author(s) : Zhang M , Chekan JR , Dodd D , Hong PY , Radlinski L , Revindran V , Nair SK , Mackie RI , Cann I
Ref : Proc Natl Acad Sci U S A , 111 :E3708 , 2014
Abstract : Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 A), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 A), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 A) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.
ESTHER : Zhang_2014_Proc.Natl.Acad.Sci.U.S.A_111_E3708
PubMedSearch : Zhang_2014_Proc.Natl.Acad.Sci.U.S.A_111_E3708
PubMedID: 25136124

Title : Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis - Agarwal_2012_Proc.Natl.Acad.Sci.U.S.A_109_17406
Author(s) : Agarwal V , Lin S , Lukk T , Nair SK , Cronan JE
Ref : Proc Natl Acad Sci U S A , 109 :17406 , 2012
Abstract : Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 alpha,omega-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-A resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.
ESTHER : Agarwal_2012_Proc.Natl.Acad.Sci.U.S.A_109_17406
PubMedSearch : Agarwal_2012_Proc.Natl.Acad.Sci.U.S.A_109_17406
PubMedID: 23045647
Gene_locus related to this paper: ecoli-bioh

Title : Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals - Gonzalez-Gutierrez_2012_Proc.Natl.Acad.Sci.U.S.A_109_6331
Author(s) : Gonzalez-Gutierrez G , Lukk T , Agarwal V , Papke D , Nair SK , Grosman C
Ref : Proc Natl Acad Sci U S A , 109 :6331 , 2012
Abstract : The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.
ESTHER : Gonzalez-Gutierrez_2012_Proc.Natl.Acad.Sci.U.S.A_109_6331
PubMedSearch : Gonzalez-Gutierrez_2012_Proc.Natl.Acad.Sci.U.S.A_109_6331
PubMedID: 22474383