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Eric D. Brown - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-Ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.
Journal of structural biology, 2021Co-Authors: Robert T. Gale, Eric D. Brown, Evgeniy V. Petrotchenko, Christoph H. Borchers, Natalie C. J. StrynadkaAbstract:Abstract The cell wall of many pathogenic Gram-positive bacteria contains Ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-Ribitol, the activated precursor for Ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-Ribitol synthesis from CTP and Ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to Ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-Ribitol production that may aid in the design of inhibitors against WTA biosynthesis.
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Duplication of teichoic acid biosynthetic genes in Staphylococcus aureus leads to functionally redundant poly(Ribitol phosphate) polymerases
Journal of bacteriology, 2008Co-Authors: Mark P. Pereira, Michael A. D'elia, Justyna Troczynska, Eric D. BrownAbstract:Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-Ribitol synthesis (tarI′J′ and tarIJ) as well as poly(Ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative Ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-Ribitol synthesis are also duplicated, a null mutant in only one of these (tarI′J′) could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI′J′ is due to poor translation of the TarJ′ enzyme, which catalyzes the rate-limiting step in CDP-Ribitol formation. This work provides new insights into understanding the complex synthetic steps of the Ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.
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Bifunctional catalysis by CDP-Ribitol synthase: convergent recruitment of reductase and cytidylyltransferase activities in Haemophilus influenzae and Staphylococcus aureus.
Biochemistry, 2004Co-Authors: Mark P. Pereira, Eric D. BrownAbstract:CDP-Ribitol synthase catalyzes the formation of CDP-Ribitol from ribulose 5-phosphate, NADPH, and CTP. CDP-Ribitol is an activated precursor for the synthesis of virulence-associated polysaccharides in the capsule of the Gram-negative pathogen Haemophilus influenzae and in the cell walls of Gram-positive pathogens including Staphylococcus aureus. We showed previously that CDP-Ribitol synthase activity in H. influenzae is catalyzed by the bifunctional enzyme Bcs1 in a two-step reaction with reduction preceding cytidylyl transfer [Zolli, M., et al. (2001) Biochemistry 40, 5041−5048]. In the work reported here, we predicted a CDP-Ribitol synthesis locus in S. aureus tandemly arranged as tarI, encoding an orthologue of the cytidylyltransferase domain of Bcs1, and tarJ, coding for an analogue of the reductase domain of Bcs1. We have shown the formation of a functional CDP-Ribitol synthase complex between TarI and TarJ. Steady-state mechanistic studies of the CDP-Ribitol synthases TarIJ and Bcs1 revealed that t...
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Reduction precedes cytidylyl transfer without substrate channeling in distinct active sites of the bifunctional CDP-Ribitol synthase from Haemophilus influenzae.
Biochemistry, 2001Co-Authors: Michaela Zolli, Daniel J. Kobric, Eric D. BrownAbstract:CDP-Ribitol synthase is a bifunctional reductase and cytidylyltransferase that catalyzes the transformation of d-ribulose 5-phosphate, NADPH, and CTP to CDP-Ribitol, a repeating unit present in the virulence-associated polysaccharide capsules of Haemophilus influenzae types a and b [Follens, A., et al. (1999) J. Bacteriol. 181, 2001]. In the work described here, we investigated the order of the reactions catalyzed by CDP-Ribitol synthase and conducted experiments to resolve the question of substrate channeling in this bifunctional enzyme. It was determined that the synthase first catalyzed the reduction of d-ribulose 5-phosphate followed by cytidylyl transfer to d-Ribitol 5-phosphate. Steady state kinetic measurements revealed a 650-fold kinetic preference for cytidylyl transfer to d-Ribitol 5-phosphate over d-ribulose 5-phosphate. Rapid mixing studies indicated quick reduction of d-ribulose 5-phosphate with a lag in the cytidylyl transfer reaction, consistent with a requirement for the accumulation of Km...
John O. Cisar - One of the best experts on this subject based on the ideXlab platform.
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comparative structural and molecular characterization of Ribitol 5 phosphate containing streptococcus oralis coaggregation receptor polysaccharides
Journal of Bacteriology, 2009Co-Authors: Jinghua Yang, Yasuo Yoshida, Mary Ritchey, Allen C Bush, John O. CisarAbstract:The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2Ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and Ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different Ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.
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Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides
Journal of bacteriology, 2009Co-Authors: Jinghua Yang, Yasuo Yoshida, Mary Ritchey, C. Allen Bush, John O. CisarAbstract:The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2Ribitol-5-PO4−-6Galfβ1-3Galβ1]n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and Ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different Ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.
Hee-seung Lee - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid
Organic letters, 2018Co-Authors: Yoon-chul Jung, Jae-hyeok Lee, Sang Ah Kim, Timo Schmidt, Wonchul Lee, Bok Luel Lee, Hee-seung LeeAbstract:A systematically designed and synthesized Ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of Ribitol phosphate tetramers, decorated with d-alanine and N-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model.
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Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid
2018Co-Authors: Yoon-chul Jung, Jae-hyeok Lee, Sang Ah Kim, Timo Schmidt, Wonchul Lee, Bok Luel Lee, Hee-seung LeeAbstract:A systematically designed and synthesized Ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of Ribitol phosphate tetramers, decorated with d-alanine and N-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model
Jinghua Yang - One of the best experts on this subject based on the ideXlab platform.
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comparative structural and molecular characterization of Ribitol 5 phosphate containing streptococcus oralis coaggregation receptor polysaccharides
Journal of Bacteriology, 2009Co-Authors: Jinghua Yang, Yasuo Yoshida, Mary Ritchey, Allen C Bush, John O. CisarAbstract:The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2Ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and Ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different Ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.
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Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides
Journal of bacteriology, 2009Co-Authors: Jinghua Yang, Yasuo Yoshida, Mary Ritchey, C. Allen Bush, John O. CisarAbstract:The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2Ribitol-5-PO4−-6Galfβ1-3Galβ1]n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and Ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different Ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.
Yoon-chul Jung - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid
Organic letters, 2018Co-Authors: Yoon-chul Jung, Jae-hyeok Lee, Sang Ah Kim, Timo Schmidt, Wonchul Lee, Bok Luel Lee, Hee-seung LeeAbstract:A systematically designed and synthesized Ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of Ribitol phosphate tetramers, decorated with d-alanine and N-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model.
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Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid
2018Co-Authors: Yoon-chul Jung, Jae-hyeok Lee, Sang Ah Kim, Timo Schmidt, Wonchul Lee, Bok Luel Lee, Hee-seung LeeAbstract:A systematically designed and synthesized Ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of Ribitol phosphate tetramers, decorated with d-alanine and N-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model
