Structure–function characterization of cellulose synthase: relationship to other glycosyltransferases

Saxena, Inder M.; Brown, R. Malcolm; Dandekar, Thomas

doi:10.1016/s0031-9422(01)00048-6

Cited by 105 publications

(91 citation statements)

References 48 publications

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“…2; Table 2). The substitutions D236Y, D333R, Q369M and R372A within this motif in the AcsAB cellulose synthase of G. xylinus impaired the enzymic activity in vitro (Saxena & Brown, 1997;Saxena et al, 2001). Consequently, we propose that the substitutions in HmsR at similar positions may abolish its enzymic activity in poly-N-acetylglucosamine production.…”

Section: Discussionmentioning

confidence: 86%

Identification of critical amino acid residues in the plague biofilm Hms proteins

et al. 2006

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Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

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Section: Discussionmentioning

confidence: 86%

Identification of critical amino acid residues in the plague biofilm Hms proteins

et al. 2006

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“…4A) and includes aspartate residues conserved in this family. Many enzymes in this family, including BRE-3, also contain a characteristic QRXRW motif that is important for transferase function (16). Based on sequencing mutant alleles, Cry5B resistance is the null phenotype for bre-3 since several known alleles (ye9 premature stop; ye28 internal deletion) are predicted to eliminate protein function.…”

Section: Bre-3 Encodes the C Elegans Homologue Of Drosophila Eggheadmentioning

confidence: 99%

Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for Toxin-Host Interactions

Griffitts

Huffman

Whitacre

et al. 2003

Journal of Biological Chemistry

119

104

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Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

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“…The enzymes that share structural topology with Alg8 are responsible for the synthesis of large polymers and contain active sites located in the cytoplasm. In vitro activity assays have been used to demonstrate the activity of GTs such as cellulose synthase in Gluconacetobacter xylinus and Cps3S in Streptococcus pneumoniae using their respective UDP-linked sugars (Forsee et al, 2000;Saxena et al, 2001). Unfortunately, the substrate of Alg8, GDP-mannuronate, is not commercially available.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, although D296 and L336 are predicted to be involved in substrate binding and catalysis of Alg8, they appear to be involved in the stability of the protein as well. However, substitutions in the structurally aligned amino acids in cellulose synthase were not reported to affect the stability of those mutant proteins (Saxena et al, 2001). It was also interesting that domain B of Alg8 does not have a true 'QxxRW' motif, but instead has a leucine instead of the glutamine.…”

Section: Discussionmentioning

confidence: 99%

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

2008

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Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for bGTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for cdi-GMP [bis-(39,59)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.

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Structure–function characterization of cellulose synthase: relationship to other glycosyltransferases

Cited by 105 publications

References 48 publications

Identification of critical amino acid residues in the plague biofilm Hms proteins

Identification of critical amino acid residues in the plague biofilm Hms proteins

Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for Toxin-Host Interactions

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

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