Length Specificity and Polymerization Mechanism of (1,3)-β-d -Glucan Synthase in Fungal Cell Wall Biosynthesis
| Autor: | J. Andrew Alspaugh, Abhishek Chhetri, Mi Jung Kim, Anna Loksztejn, Kaila M. Pianalto, Hai P Nguyen, Kenichi Yokoyama, Jiyong Hong |
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| Rok vydání: | 2020 |
| Předmět: |
beta-Glucans
Protein subunit Saccharomyces cerevisiae Degree of polymerization Biochemistry Article Polymerization 03 medical and health sciences Species Specificity Cell Wall Glucan chemistry.chemical_classification 0303 health sciences ATP synthase biology Chemistry Antifungal antibiotic 030302 biochemistry & molecular biology Substrate (chemistry) Yeast Kinetics Glucosyltransferases Biocatalysis Chromatography Gel biology.protein Proteoglycans |
| Zdroj: | Biochemistry. 59:682-693 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00896 |
| Popis: | (1,3)-β-D-Glucan synthase (GS) catalyzes formation of the linear (1,3)-β-D-glucan in the fungal cell wall, and is a target of clinically approved antifungal antibiotics. The catalytic subunit of GS, FKS protein, does not exhibit significant sequence homologies to other glycosyltransferases, and thus a significant ambiguity remains about its catalytic mechanism. One of the major technical barriers in studying GS is the absence of activity assay methods that allow characterization of the lengths and amounts of (1,3)-β-D-glucan due to its poor solubility in water and organic solvents. Here, we report a successful development of a novel GS activity assay based on size-exclusion chromatography coupled with pulsed-amperometric detection and radiation counting (SEC-PAD-RC), which allows for the simultaneous characterization of the amount and length of the polymer product. The assay revealed that the purified yeast GS produces glucan with a length of 6,550 ± 760 mer, consistent with the reported degree of polymerization of (1,3)-β-D-glucan isolated from intact cells. Pre-steady state kinetics analysis revealed a highly efficient but rate-determining chain elongation rate of 51.5 ± 9.8 sec(−1), which represents the first observation of chain elongation by a nucleotide-sugar dependent polysaccharide synthase. Coupling the SEC-PAD-RC method with substrate analog mechanistic probes provided the first unambiguous evidence that GS catalyzes non-reducing end polymerization. Based on these observations, we propose a detailed model for the catalytic mechanism of GS. The approaches described here can be used to determine the mechanism of catalysis of other polysaccharide synthases. |
| Databáze: | OpenAIRE |
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