Browsing by Author "Wang, Fengbin"
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Item 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.Item Developing an Enzyme Toolbox for Anti-Tumor Natural Product Biosynthesis(2016-04-21) Wang, Fengbin; Phillips, Jr., George N.; Nicolaou, Kyriacos C.Natural products, known as chemical compounds or substances produced by living organisms, are arguably the most important players in revolutionizing modern medicine. To date, natural products remain the best source of drug leads, including penicillin, streptomycin, artemisinin and others. However, one of the difficulties of natural product-based drug discovery is the structural derivatization. The traditional chemical modification methods often require multiple protection and deprotection steps for the large variety of functional groups, making the process laborious and problematic. A promising alternative approach to produce natural product derivatives is utilizing biosynthesis enzyme toolbox; for example, enzyme-based "glycol-randomization" and "alkyl-randomization" can dramatically expand diversities of important anti-cancer natural products. In my thesis work, I characterized seven biosynthesis enzymes (including sugar aminotransferase WecE, CalS13, AtmS13, glycosyltransferase SsfS6, OleD, methionine adenosyltransferase sMAT and methyltransferase DnrK) by X-ray crystallography. The structural characterizations and protein engineering of those enzymes lead to successfully expand the glyco/alkyl libraries, as well as to broaden the glyco/alkyl installation processes. These discoveries and enzyme toolbox developments are directly applicable to future drug discovery for cancer, and can be utilized as blue print to further understand the essential role of glycosylation and methylation in biology.Item Reader domain specificity and lysine demethylase-4 family function(Springer Nature, 2016) Su, Zhangli; Wang, Fengbin; Lee, Jin-Hee; Stephens, Kimberly E.; Papazyan, Romeo; Voronina, Ekaterina; Krautkramer, Kimberly A.; Raman, Ana; Thorpe, Jeremy J.; Boersma, Melissa D.; Kuznetsov, Vyacheslav I.; Miller, Mitchell D.; Taverna, Sean D.; Phillips, George N.Jr.; Denu, John M.The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitrodemethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis.Item 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.Item 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.Item 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.Item Structure of a cupin protein Plu4264 from Photorhabdus luminescens subsp. laumondii TTO1 at 1.35 Å resolution(Wiley, 2015) Weerth, R.Sophia; Michalska, Karolina; Bingman, Craig A.; Yennamalli, Ragothaman M.; Li, Hui; Jedrzejczak, Robert; Wang, Fengbin; Babnigg, Gyorgy; Joachimiak, Andrzej; Thomas, Michael G.; Phillips, George N.Jr.Proteins belonging to the cupin superfamily have a wide range of catalytic and noncatalytic functions. Cupin proteins commonly have the capacity to bind a metal ion with the metal frequently determining the function of the protein. We have been investigating the function of homologous cupin proteins that are conserved in more than 40 species of bacteria. To gain insights into the potential function of these proteins we have solved the structure of Plu4264 from Photorhabdus luminescens TTO1 at a resolution of 1.35 Å and identified manganese as the likely natural metal ligand of the protein.Item 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.Item 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.