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Daniel Wefers - One of the best experts on this subject based on the ideXlab platform.

  • Detailed Structural Characterization of Arabinans and Galactans of 14 Apple Cultivars Before and After Cold Storage.
    Frontiers in Plant Science, 2018
    Co-Authors: Daniel Wefers, Ramona Flörchinger, Mirko Bunzel
    Abstract:

    Physiological and textural properties of apples are greatly influenced by both cultivar and structural composition of their pectic polysaccharides. In previous studies it was demonstrated that neutral pectic side chains (Arabinans and galactans) play a major role during fruit development and postharvest processes. However, these complex polymers have a high structural heterogeneity, and some structural elements such as side chain substituents and substitution of neighboring residues cannot be analyzed by using conventional analytical methods. Therefore, fine structures of Arabinans and galactans were analyzed in 14 apple cultivars before and after storage. Besides conventional methods such as methylation analysis, profiling approaches based on enzymatic cleavage were applied to obtain detailed information on the neutral side chains of pectins. Structurally different, highly branched Arabinans and linear β-1,4-linked galactans were detected in all cultivars. By using enzymatic profiling approaches, rare structural elements such as β-arabinofuranose and α-arabinopyranose residues were detected. In addition, the combination of all methods indicated structural differences with regard to ramification position or patterns. Cold storage resulted in decreased portions of branched Arabinans. It was demonstrated that arabinan decomposition is independent of previously detected structural variations. In addition, analysis of endo-arabinanase hydrolysates demonstrated that β-arabinofuranose containing side chains are enriched after storage and may play a major role in postharvest processes. Analysis of endo galactanase hydrolysates showed decreased portions of galactan-bound, terminal α-arabinopyranose units after storage. Therefore, these residues are most likely removed during postharvest galactan decomposition. The results of this study demonstrate the high complexity of neutral pectin side chains in apples and that pectic structural elements are differently prone to postharvest modifications.

  • Data_Sheet_1_Detailed Structural Characterization of Arabinans and Galactans of 14 Apple Cultivars Before and After Cold Storage.pdf
    2018
    Co-Authors: Daniel Wefers, Ramona Flörchinger, Mirko Bunzel
    Abstract:

    Physiological and textural properties of apples are greatly influenced by both cultivar and structural composition of their pectic polysaccharides. In previous studies, it was demonstrated that neutral pectic side chains (Arabinans and galactans) play a major role during fruit development and postharvest processes. However, these complex polymers have a high structural heterogeneity, and some structural elements such as side chain substituents and substitution of neighboring residues cannot be analyzed by using conventional analytical methods. Therefore, fine structures of Arabinans and galactans were analyzed in 14 apple cultivars before and after storage. Besides conventional methods such as methylation analysis, profiling approaches based on enzymatic cleavage were applied to obtain detailed information on the neutral side chains of pectins. Structurally different, highly branched Arabinans and linear β-1,4-linked galactans were detected in all cultivars. By using enzymatic profiling approaches, rare structural elements such as β-arabinofuranose and α-arabinopyranose residues were detected. In addition, the combination of all methods indicated structural differences with regard to ramification position or patterns. Cold storage resulted in decreased portions of branched Arabinans. It was demonstrated that arabinan decomposition is independent of previously detected structural variations. In addition, analysis of endo-arabinanase hydrolysates demonstrated that β-arabinofuranose containing side chains are enriched after storage and may play a major role in postharvest processes. Analysis of endo-galactanase hydrolysates showed decreased portions of galactan-bound, terminal α-arabinopyranose units after storage. Therefore, these residues are most likely removed during postharvest galactan decomposition. The results of this study demonstrate the high complexity of neutral pectin side chains in apples and that pectic structural elements are differently prone to postharvest modifications.

  • Enzymatic mechanism for arabinan degradation and transport in the thermophilic bacterium Caldanaerobius polysaccharolyticus
    Applied and environmental microbiology, 2017
    Co-Authors: Daniel Wefers, Jia Dong, Ahmed M. Abdel-hamid, Hans Müller Paul, Gabriel V. Pereira, Yejun Han, Dylan Dodd, Ramiya Baskaran, Beth Mayer, Roderick I. Mackie
    Abstract:

    The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusIMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of Arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

  • Enzymatic mechanism for arabinan degradation and transport in the thermophilic bacterium Caldanaerobius polysaccharolyticus
    Applied and Environmental Microbiology, 2017
    Co-Authors: Daniel Wefers, Jia Dong, Ahmed M. Abdel-hamid, Hans Müller Paul, Gabriel V. Pereira, Yejun Han, Dylan Dodd, Ramiya Baskaran, Beth Mayer, Roderick I. Mackie
    Abstract:

    The plant cell wall polysaccharide arabinan is an important supply of arabinose, and unraveling arabinan degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-L-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endo-arabinanase (CpAbn43A), a GH27 β-L-arabinopyranosidase (CpAbp27A), and two GH127 β-L-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with p NP-linked sugars and naturally occurring pectin structural elements as substrates. The results demonstrated that CpAbn43A is an endo-arabinanase, while CpAbf51A and CpAbf51B are α-L-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2-, α-1,3-, and α-1,5-linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60 °C and 75 °C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endo-arabinanase and L-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus . Importance Genomic DNA sequencing and bioinformatic analysis allowed for the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus . The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided detailed understanding of the substrate specificities of the enzymes, and allowed assignment of two new GH127 polypeptides as β-L-arabinofuranosidases, able to degrade pectic substrates, and thus expanding our knowledge on this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of Arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

  • NMR Spectroscopic Profiling of Arabinan and Galactan Structural Elements
    Journal of agricultural and food chemistry, 2016
    Co-Authors: Daniel Wefers, Mirko Bunzel
    Abstract:

    Pectic Arabinans and galactans presumably affect the physiological and technological properties of plant cell walls and dietary fiber. Their complex structures are usually analyzed by time-consuming methods, which are based on chemical cleavage to monomers. To gain more detailed insights into the arabinan and galactan structures, a time-efficient approach based on enzymatic cleavage and two-dimensional NMR spectroscopy was developed. Heteronuclear single quantum coherence spectroscopy (HSQC) marker signals were evaluated for various structural elements, and relative response factors were determined, allowing a semiquantitative estimation of the structural composition. The method was applied to analyze different insoluble plant materials and soluble polysaccharides. It was demonstrated that the developed approach yielded comparable information about various structural elements that can also be detected by using the conventional methylation analysis. However, by using the NMR method, additional structural i...

Mirko Bunzel - One of the best experts on this subject based on the ideXlab platform.

  • Detailed Structural Characterization of Arabinans and Galactans of 14 Apple Cultivars Before and After Cold Storage.
    Frontiers in Plant Science, 2018
    Co-Authors: Daniel Wefers, Ramona Flörchinger, Mirko Bunzel
    Abstract:

    Physiological and textural properties of apples are greatly influenced by both cultivar and structural composition of their pectic polysaccharides. In previous studies it was demonstrated that neutral pectic side chains (Arabinans and galactans) play a major role during fruit development and postharvest processes. However, these complex polymers have a high structural heterogeneity, and some structural elements such as side chain substituents and substitution of neighboring residues cannot be analyzed by using conventional analytical methods. Therefore, fine structures of Arabinans and galactans were analyzed in 14 apple cultivars before and after storage. Besides conventional methods such as methylation analysis, profiling approaches based on enzymatic cleavage were applied to obtain detailed information on the neutral side chains of pectins. Structurally different, highly branched Arabinans and linear β-1,4-linked galactans were detected in all cultivars. By using enzymatic profiling approaches, rare structural elements such as β-arabinofuranose and α-arabinopyranose residues were detected. In addition, the combination of all methods indicated structural differences with regard to ramification position or patterns. Cold storage resulted in decreased portions of branched Arabinans. It was demonstrated that arabinan decomposition is independent of previously detected structural variations. In addition, analysis of endo-arabinanase hydrolysates demonstrated that β-arabinofuranose containing side chains are enriched after storage and may play a major role in postharvest processes. Analysis of endo galactanase hydrolysates showed decreased portions of galactan-bound, terminal α-arabinopyranose units after storage. Therefore, these residues are most likely removed during postharvest galactan decomposition. The results of this study demonstrate the high complexity of neutral pectin side chains in apples and that pectic structural elements are differently prone to postharvest modifications.

  • Data_Sheet_1_Detailed Structural Characterization of Arabinans and Galactans of 14 Apple Cultivars Before and After Cold Storage.pdf
    2018
    Co-Authors: Daniel Wefers, Ramona Flörchinger, Mirko Bunzel
    Abstract:

    Physiological and textural properties of apples are greatly influenced by both cultivar and structural composition of their pectic polysaccharides. In previous studies, it was demonstrated that neutral pectic side chains (Arabinans and galactans) play a major role during fruit development and postharvest processes. However, these complex polymers have a high structural heterogeneity, and some structural elements such as side chain substituents and substitution of neighboring residues cannot be analyzed by using conventional analytical methods. Therefore, fine structures of Arabinans and galactans were analyzed in 14 apple cultivars before and after storage. Besides conventional methods such as methylation analysis, profiling approaches based on enzymatic cleavage were applied to obtain detailed information on the neutral side chains of pectins. Structurally different, highly branched Arabinans and linear β-1,4-linked galactans were detected in all cultivars. By using enzymatic profiling approaches, rare structural elements such as β-arabinofuranose and α-arabinopyranose residues were detected. In addition, the combination of all methods indicated structural differences with regard to ramification position or patterns. Cold storage resulted in decreased portions of branched Arabinans. It was demonstrated that arabinan decomposition is independent of previously detected structural variations. In addition, analysis of endo-arabinanase hydrolysates demonstrated that β-arabinofuranose containing side chains are enriched after storage and may play a major role in postharvest processes. Analysis of endo-galactanase hydrolysates showed decreased portions of galactan-bound, terminal α-arabinopyranose units after storage. Therefore, these residues are most likely removed during postharvest galactan decomposition. The results of this study demonstrate the high complexity of neutral pectin side chains in apples and that pectic structural elements are differently prone to postharvest modifications.

  • NMR Spectroscopic Profiling of Arabinan and Galactan Structural Elements
    Journal of agricultural and food chemistry, 2016
    Co-Authors: Daniel Wefers, Mirko Bunzel
    Abstract:

    Pectic Arabinans and galactans presumably affect the physiological and technological properties of plant cell walls and dietary fiber. Their complex structures are usually analyzed by time-consuming methods, which are based on chemical cleavage to monomers. To gain more detailed insights into the arabinan and galactan structures, a time-efficient approach based on enzymatic cleavage and two-dimensional NMR spectroscopy was developed. Heteronuclear single quantum coherence spectroscopy (HSQC) marker signals were evaluated for various structural elements, and relative response factors were determined, allowing a semiquantitative estimation of the structural composition. The method was applied to analyze different insoluble plant materials and soluble polysaccharides. It was demonstrated that the developed approach yielded comparable information about various structural elements that can also be detected by using the conventional methylation analysis. However, by using the NMR method, additional structural i...

  • Arabinan and Galactan Oligosaccharide Profiling by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).
    Journal of agricultural and food chemistry, 2016
    Co-Authors: Daniel Wefers, Mirko Bunzel
    Abstract:

    Arabinans and galactans are complex pectic polysaccharides, which greatly influence the physicochemical and physiological properties of plants and plant-based foods. Conventional methods to characterize these challenging polymers are based on derivatization and/or unselective chemical cleavage of the glycosidic bonds of the polysaccharides, resulting in partial loss of essential information such as anomeric configuration. Here, endo-arabinanase and endo-galactanase were used to selectively cleave pectic Arabinans and galactans. The liberated oligosaccharides were purified and characterized by LC-MS and one- and two-dimensional NMR spectroscopy resulting in known but also several previously unknown pectic structural elements. For the routine analysis of pectin hydrolysates by HPAEC-PAD, incubation conditions, chromatographic parameters, and relative response factors of the isolated pectic oligosaccharides against an internal standard were determined. The applicability of the method was demonstrated by anal...

  • Arabinan and Galactan Oligosaccharide Profiling by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)
    2016
    Co-Authors: Daniel Wefers, Mirko Bunzel
    Abstract:

    Arabinans and galactans are complex pectic polysaccharides, which greatly influence the physicochemical and physiological properties of plants and plant-based foods. Conventional methods to characterize these challenging polymers are based on derivatization and/or unselective chemical cleavage of the glycosidic bonds of the polysaccharides, resulting in partial loss of essential information such as anomeric configuration. Here, endo-arabinanase and endo-galactanase were used to selectively cleave pectic Arabinans and galactans. The liberated oligosaccharides were purified and characterized by LC-MS and one- and two-dimensional NMR spectroscopy resulting in known but also several previously unknown pectic structural elements. For the routine analysis of pectin hydrolysates by HPAEC-PAD, incubation conditions, chromatographic parameters, and relative response factors of the isolated pectic oligosaccharides against an internal standard were determined. The applicability of the method was demonstrated by analyzing different well-characterized plant cell wall materials. It was demonstrated that the developed method yields additional information about pectic arabinan and galactan structures that is not obtained from conventional methods such as methylation analysis

Michael R Mcneil - One of the best experts on this subject based on the ideXlab platform.

  • A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.
    The Journal of biological chemistry, 2014
    Co-Authors: Devinder Kaur, Kay-hooi Khoo, Patrick J. Brennan, Delphi Chatterjee, Shiva K. Angala, Mary Jackson, Michael R Mcneil
    Abstract:

    Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of Arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

  • the identification and location of succinyl residues and the characterization of the interior arabinan region allow for a model of the complete primary structure of mycobacterium tuberculosis mycolyl arabinogalactan
    Journal of Biological Chemistry, 2008
    Co-Authors: Suresh Bhamidi, Kay-hooi Khoo, Delphi Chatterjee, Michael S. Scherman, Christopher D Rithner, Jessica E Prenni, Michael R Mcneil
    Abstract:

    The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-α-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear α-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

  • the carboxy terminus of embc from mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan
    Journal of Biological Chemistry, 2006
    Co-Authors: Stefan Berg, Kay-hooi Khoo, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Delphi Chatterjee
    Abstract:

    Abstract d-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, β-d-Araf(1→2)-α-d-Araf(1→5)-α-d-Araf(1→5)-α-d-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-d-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • The Carboxy Terminus of EmbC from Mycobacterium smegmatis Mediates Chain Length Extension of the Arabinan in Lipoarabinomannan
    The Journal of biological chemistry, 2006
    Co-Authors: Libin Shi, Kay-hooi Khoo, Stefan Berg, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Arwen Lee, Delphi Chatterjee
    Abstract:

    D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • Development of a Quantitative Assay for Mycobacterial Endogenous Arabinase and Ensuing Studies of Arabinase Levels and Arabinan Metabolism in Mycobacterium smegmatis
    Applied and environmental microbiology, 2006
    Co-Authors: Xu Dong, Suresh Bhamidi, Michael S. Scherman, Yi Xin, Michael R Mcneil
    Abstract:

    Treatment of either Mycobacterium tuberculosis or M. smegmatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [14C]glucose, followed by a “chase” with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.

Delphi Chatterjee - One of the best experts on this subject based on the ideXlab platform.

  • A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.
    The Journal of biological chemistry, 2014
    Co-Authors: Devinder Kaur, Kay-hooi Khoo, Patrick J. Brennan, Delphi Chatterjee, Shiva K. Angala, Mary Jackson, Michael R Mcneil
    Abstract:

    Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of Arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

  • the identification and location of succinyl residues and the characterization of the interior arabinan region allow for a model of the complete primary structure of mycobacterium tuberculosis mycolyl arabinogalactan
    Journal of Biological Chemistry, 2008
    Co-Authors: Suresh Bhamidi, Kay-hooi Khoo, Delphi Chatterjee, Michael S. Scherman, Christopher D Rithner, Jessica E Prenni, Michael R Mcneil
    Abstract:

    The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-α-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear α-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

  • the carboxy terminus of embc from mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan
    Journal of Biological Chemistry, 2006
    Co-Authors: Stefan Berg, Kay-hooi Khoo, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Delphi Chatterjee
    Abstract:

    Abstract d-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, β-d-Araf(1→2)-α-d-Araf(1→5)-α-d-Araf(1→5)-α-d-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-d-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • The Carboxy Terminus of EmbC from Mycobacterium smegmatis Mediates Chain Length Extension of the Arabinan in Lipoarabinomannan
    The Journal of biological chemistry, 2006
    Co-Authors: Libin Shi, Kay-hooi Khoo, Stefan Berg, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Arwen Lee, Delphi Chatterjee
    Abstract:

    D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • Truncated Structural Variants of Lipoarabinomannan in Ethambutol Drug-resistant Strains of Mycobacterium smegmatis INHIBITION OF ARABINAN BIOSYNTHESIS BY ETHAMBUTOL
    The Journal of biological chemistry, 1996
    Co-Authors: Kay-hooi Khoo, Edward Douglas, Parastoo Azadi, Julia M. Inamine, Gurdyal S. Besra, Katarína Mikušová, Patrick J. Brennan, Delphi Chatterjee
    Abstract:

    The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of Arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 microg/ml Emb (Mikusova, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of Arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.

Kay-hooi Khoo - One of the best experts on this subject based on the ideXlab platform.

  • A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.
    The Journal of biological chemistry, 2014
    Co-Authors: Devinder Kaur, Kay-hooi Khoo, Patrick J. Brennan, Delphi Chatterjee, Shiva K. Angala, Mary Jackson, Michael R Mcneil
    Abstract:

    Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of Arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

  • the identification and location of succinyl residues and the characterization of the interior arabinan region allow for a model of the complete primary structure of mycobacterium tuberculosis mycolyl arabinogalactan
    Journal of Biological Chemistry, 2008
    Co-Authors: Suresh Bhamidi, Kay-hooi Khoo, Delphi Chatterjee, Michael S. Scherman, Christopher D Rithner, Jessica E Prenni, Michael R Mcneil
    Abstract:

    The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-α-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear α-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

  • the carboxy terminus of embc from mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan
    Journal of Biological Chemistry, 2006
    Co-Authors: Stefan Berg, Kay-hooi Khoo, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Delphi Chatterjee
    Abstract:

    Abstract d-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, β-d-Araf(1→2)-α-d-Araf(1→5)-α-d-Araf(1→5)-α-d-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-d-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • The Carboxy Terminus of EmbC from Mycobacterium smegmatis Mediates Chain Length Extension of the Arabinan in Lipoarabinomannan
    The Journal of biological chemistry, 2006
    Co-Authors: Libin Shi, Kay-hooi Khoo, Stefan Berg, John S Spencer, Jian Zhang, Varalakshmi D Vissa, Michael R Mcneil, Arwen Lee, Delphi Chatterjee
    Abstract:

    D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the Arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller Arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

  • Characterization of the in vitro synthesized arabinan of mycobacterial cell walls.
    Biochimica et biophysica acta, 1997
    Co-Authors: Yi Xin, Kay-hooi Khoo, Gurdyal S. Besra, Patrick J. Brennan, Richard E. Lee, Michael S. Scherman, Michael R Mcneil
    Abstract:

    Abstract Previous studies have shown that polymerized [14C]arabinan can be synthesized from polyprenylphosphate-[14C]arabinose by the particulate enzymes of Mycobacterium smegmatis [R.E. Lee, K. Mikusova, P.J. Brennan and G.S Besra (1995) J. Am. Chem. Soc. 117, 11829–11832]. In the present investigation, the [14C]arabinan product was biochemically characterized. Sizing chromatography revealed a molecular weight consistent with that expected from mature arabinan. Digestion of the [14C]arabinan with a mixture of arabinases produced oligo[14C]arabinoside fragments including hexa[14C]arabinoside and tetra[14C]arabinoside which originated from the non-reducing terminal regions of the polymer, and di[14C]arabinoside from the internal regions of the polymer. These arabinoside fragments represent the major known structural motifs that comprise the arabinan segment of arabinogalactan and lipoarabinomannan. The presence of [14C]arabinose in both the internal and external regions of the [14C]arabinan suggests that polyprenylphosphate-arabinose is the major, and perhaps the only, donor of arabinosyl residues in mycobacteria.