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Xi Chen - One of the best experts on this subject based on the ideXlab platform.
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a sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating lewisx
ACS Chemical Biology, 2012Co-Authors: Go Sugiarto, Kam Lau, Sunghyuk Lim, James B Ames, Andrew J Fisher, Xi ChenAbstract:Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a Protein Crystal Structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant Structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.
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pasteurella multocida cmp sialic acid synthetase and mutants of neisseria meningitidis cmp sialic acid synthetase with improved substrate promiscuity
Applied Microbiology and Biotechnology, 2012Co-Authors: Yanhong Li, Saddam Muthana, Hai Yu, Xi ChenAbstract:Cytidine 5′-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on Protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as Crystal Structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and Protein Crystal Structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Yanhong Li, Hai Yu, Zahra Khedri, Diem Thuy Le, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Kam Lau, Zahra Khedri, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
Go Sugiarto - One of the best experts on this subject based on the ideXlab platform.
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a sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating lewisx
ACS Chemical Biology, 2012Co-Authors: Go Sugiarto, Kam Lau, Sunghyuk Lim, James B Ames, Andrew J Fisher, Xi ChenAbstract:Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a Protein Crystal Structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant Structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Yanhong Li, Hai Yu, Zahra Khedri, Diem Thuy Le, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Kam Lau, Zahra Khedri, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
Marc Messerschmidt - One of the best experts on this subject based on the ideXlab platform.
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de novo Protein Crystal Structure determination from x ray free electron laser data
Nature, 2014Co-Authors: Thomas R M Barends, Lutz Foucar, Sabine Botha, Bruce R Doak, Robert L Shoeman, Karol Nass, Jason E Koglin, Garth J Williams, Sebastien Boutet, Marc MesserschmidtAbstract:The determination of Protein Crystal Structures is hampered by the need for macroscopic Crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized Crystals in a 'diffraction-before-destruction' approach. So far, all Protein Structure determinations carried out using FELs have been based on previous knowledge of related, known Structures. Here we show that X-ray FEL data can be used for de novo Protein Structure determination, that is, without previous knowledge about the Structure. Using the emerging technique of serial femtosecond Crystallography, we performed single-wavelength anomalous scattering measurements on microCrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the Protein Structure. This demonstrates the feasibility of determining novel Protein Structures using FELs. We anticipate that serial femtosecond Crystallography will become an important tool for the Structure determination of Proteins that are difficult to Crystallize, such as membrane Proteins.
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de novo Protein Crystal Structure determination from x ray free electron laser data
Nature, 2014Co-Authors: Thomas R M Barends, Lutz Foucar, Sabine Botha, Bruce R Doak, Robert L Shoeman, Karol Nass, Jason E Koglin, Garth J Williams, Sebastien Boutet, Marc MesserschmidtAbstract:Femtosecond Crystallography with an X-ray free-electron laser is used to analyse micrometre-sized Protein Crystals, generating a high-resolution Structure of the Protein without previous knowledge of what it looks like. X-ray Crystallographers typically spend a great deal of time optimizing Crystallization conditions to obtain the large, well-ordered Crystals needed to generate high-quality data sets. It was shown recently that extremely short and intense pulses of X-rays from X-ray free-electron lasers can be used to obtain diffraction data on nano-to-micrometre-sized Protein Crystals before radiation damage to the Crystal occurs. The hope is that this approach — called serial femtosecond Crystallography — will produce Structures of Proteins and Protein complexes that do not yield macroscopic, well-ordered Crystals. One of the major limitations of serial femtosecond Crystallography is that it has not been possible to solve the Structure of a Protein without prior knowledge of related, known Structures. In this paper, the authors show how serial femtosecond Crystallography with an X-ray free-electron laser can be used to experimentally solve the 'phase problem', generating a high-resolution Structure of a Protein without a priori knowledge of what the Protein looks like. The determination of Protein Crystal Structures is hampered by the need for macroscopic Crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized Crystals1,2,3,4 in a ‘diffraction-before-destruction’ approach. So far, all Protein Structure determinations carried out using FELs have been based on previous knowledge of related, known Structures1,2,3,4,5. Here we show that X-ray FEL data can be used for de novo Protein Structure determination, that is, without previous knowledge about the Structure. Using the emerging technique of serial femtosecond Crystallography1,2,3,4,6, we performed single-wavelength anomalous scattering measurements on microCrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration6,7, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the Protein Structure. This demonstrates the feasibility of determining novel Protein Structures using FELs. We anticipate that serial femtosecond Crystallography will become an important tool for the Structure determination of Proteins that are difficult to Crystallize, such as membrane Proteins1,2,8.
Yanhong Li - One of the best experts on this subject based on the ideXlab platform.
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pasteurella multocida cmp sialic acid synthetase and mutants of neisseria meningitidis cmp sialic acid synthetase with improved substrate promiscuity
Applied Microbiology and Biotechnology, 2012Co-Authors: Yanhong Li, Saddam Muthana, Hai Yu, Xi ChenAbstract:Cytidine 5′-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on Protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as Crystal Structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and Protein Crystal Structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Yanhong Li, Hai Yu, Zahra Khedri, Diem Thuy Le, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
Hai Yu - One of the best experts on this subject based on the ideXlab platform.
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pasteurella multocida cmp sialic acid synthetase and mutants of neisseria meningitidis cmp sialic acid synthetase with improved substrate promiscuity
Applied Microbiology and Biotechnology, 2012Co-Authors: Yanhong Li, Saddam Muthana, Hai Yu, Xi ChenAbstract:Cytidine 5′-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on Protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as Crystal Structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and Protein Crystal Structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 sialyltransferase 1 pmst1 by site directed mutagenesis
Molecular BioSystems, 2011Co-Authors: Go Sugiarto, Yanhong Li, Hai Yu, Zahra Khedri, Diem Thuy Le, Xi ChenAbstract:Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by Protein Crystal Structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
