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1. Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During In vitro Reactions

   https://doi.org/10.1093/pcp/pcab114  

   Ehrlich, Jacqueline J; Weerts, Richard M; Shome, Sayane; Culbertson, Alan T; Honzatko, Richard B; Jernigan, Robert L; Zabotina, Olga A (July 2021, Plant and Cell Physiology)

   null (Ed.)

   Abstract Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins, and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate Xyloglucan Xylosyltransferase 1 (XXT1), a GT involved in synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from UDP-Xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-Xylose and UDP-Glucose, while reactions with UDP-Arabinose and UDP-Galactose are over 10-fold slower. Using kcat/Km as a measure of efficiency, UDP-Xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-Glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific, and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems. 

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2. Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-glucan Synthase that Functions during Plant Cell Wall Synthesis.

   https://doi.org/doi: 10.1105/tpc.19.00637  

   *Yang J., *Bak G. (May 2020, The plant cell)

   In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6. 

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3. The Arabidopsis xylosyltransferases, XXT3 , XXT4 , and XXT5, are essential to complete the fully xylosylated glucan backbone XXXG ‐type structure of xyloglucans

   https://doi.org/10.1111/nph.18851  

   Zhang, Ning; Julian, Jordan D.; Yap, Cheng Ern; Swaminathan, Sivakumar; Zabotina, Olga A. (April 2023, New Phytologist)

   Summary Although most xyloglucans (XyGs) biosynthesis enzymes have been identified, the molecular mechanism that defines XyG branching patterns is unclear. Four out of five XyG xylosyltransferases (XXT1, XXT2, XXT4, and XXT5) are known to add the xylosyl residue from UDP‐xylose onto a glucan backbone chain; however, the function of XXT3 has yet to be demonstrated.Singlexxt3and triplexxt3xxt4xxt5mutantArabidopsis(Arabidopsis thaliana) plants were generated using CRISPR‐Cas9 technology to determine the specific function of XXT3.Combined biochemical, bioinformatic, and morphological data conclusively established for the first time that XXT3, together with XXT4 and XXT5, adds xylosyl residue specifically at the third glucose in the glucan chain to synthesize XXXG‐type XyGs. We propose that the specificity of XXT3, XXT4, and XXT5 is directed toward the prior synthesis of the acceptor substrate by the other two enzymes, XXT1 and XXT2. We also conclude that XXT5 plays a dominant role in the synthesis of XXXG‐type XyGs, while XXT3 and XXT4 complementarily contribute their activities in a tissue‐specific manner.The newly generatedxxt3xxt4xxt5mutant produces only XXGG‐type XyGs, which further helps to understand the impact of structurally deficient polysaccharides on plant cell wall organization, growth, and development. 

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4. Towards Computationally Guided Design and Engineering of a Neisseria meningitidis Serogroup W Capsule Polymerase with Altered Substrate Specificity

   https://doi.org/10.3390/pr9122192  

   Paudel, Subhadra; Wachira, James; McCarthy, Pumtiwitt C. (December 2021, Processes)

   Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of target metal ions and overall binding capacity. Microorganisms are a key component in wastewater treatment plants, and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose–sialic acid (Gal–NeuNAc) heteropolysaccharide binds lead ions effectively, thereby demonstrating the potential for its use in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis of N-acetylgalactosamine–sialic acid (GalNAc–NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics (MD) simulations with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated. 

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5. Characterization of the need for galactofuranose during the Neurospora crassa life cycle

   https://doi.org/10.1016/j.fgb.2023.103826  

   Schaff, Hayden; Dey, Protyusha; Heiss, Christian; Keiser, Griffin; Moro, Tatiana Rojo; Azadi, Parastoo; Patel, Pavan; Free, Stephen J. (October 2023, Fungal Genetics and Biology)

   Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases. 

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