Browsing by Author "Singh, Shanteri"

Now showing 1 - 8 of 8
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    Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis
    (Wiley, 2013) Wang, Fengbin; Zhou, Maoquan; Singh, Shanteri; Yennamalli, Ragothaman M.; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.Jr.
    The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the CC glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.
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    Loop dynamics of thymidine diphosphate-rhamnose 3′-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis
    (AIP Publishing, 2016) Han, Lu; Singh, Shanteri; Thorson, Jon S.; Phillips, George N.Jr.
    Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3′-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3′-O-methyltransferase (CalS11) catalyses a 3′-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present thecrystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.
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    Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases
    (American Chemical Society, 2015) Wang, Fengbin; Singh, Shanteri; Xu, Weijun; Helmich, Kate E.; Miller, Mitchell D.; Cao, Hongnan; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.Jr.
    Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.
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    Structural characterization of AtmS13, a putative sugar aminotransferase involved in indolocarbazole AT2433 aminopentose biosynthesis
    (Wiley, 2015) Singh, Shanteri; Kim, Youngchang; Wang, Fengbin; Bigelow, Lance; Endres, Michael; Kharel, Madan K.; Babnigg, Gyorgy; Bingman, Craig A.; Joachimiak, Andrzej; Thorson, Jon S.; Phillips, George N.Jr.
    AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50-Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose vs. pentose) and/or sugar C2 (deoxy vs. hydroxyl) substrate specificity.
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    Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate
    (AIP Publishing LLC, 2016) Cao, Hongnan; Tan, Kemin; Wang, Fengbin; Bigelow, Lance; Yennamalli, Ragothaman M.; Jedrzejczak, Robert; Babnigg, Gyorgy; Bingman, Craig A.; Joachimiak, Andrzej; Kharel, Madan K.; Singh, Shanteri; Thorson, Jon S.; Phillips, George N.Jr.
    CalE6 from Micromonospora echinospora is a (pyridoxal 5′ phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation.
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    Structure and specificity of a permissive bacterial C-prenyltransferase
    (Springer Nature, 2017) Elshahawi, Sherif I.; Cao, Hongnan; Shaaban, Khaled A.; Ponomareva, Larissa V.; Subramanian, Thangaiah; Farman, Mark L.; Spielmann, H.Peter; Phillips, George N.Jr.; Thorson, Jon S.; Singh, Shanteri
    This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.
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    Structure-Guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19
    (American Chemical Society, 2014) Elshahawi, Sherif I.; Ramelot, Theresa A.; Seetharaman, Jayaraman; Chen, Jing Han; Singh, Shanteri; Yang, Yunhuang; Pederson, Kari; Kharel, Madan K.; Xiao, Rong; Lew, Scott; Yennamalli, Ragothaman M.; Miller, Mitchell D.; Wang, Fengbin; Tong, Liang; Montelione, Gaetano T.; Kennedy, Michael A.; Bingman, Craig A.; Zhu, Haining; Phillips, George N.Jr.; Thorson, Jon S.
    Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as “unknowns”, CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.
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    Understanding molecular recognition of promiscuity of thermophilic methionine adenosyltransferase sMAT from Sulfolobus solfataricus
    (Wiley, 2014) Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.Jr.