Length Specificity and Polymerization Mechanism of (1,3)-β-d-Glucan Synthase in Fungal Cell Wall Biosynthesis.,Biochemistry

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Length Specificity and Polymerization Mechanism of (1,3)-β-d-Glucan Synthase in Fungal Cell Wall Biosynthesis.
Biochemistry ( IF 2.9 ) Pub Date : 2020-01-03 , DOI: 10.1021/acs.biochem.9b00896
Abhishek Chhetri ₁ , Anna Loksztejn ₁ , Hai Nguyen ₁ , Kaila M Pianalto ₂ , Mi Jung Kim ₃ , Jiyong Hong ₃ , J Andrew Alspaugh ₂ , Kenichi Yokoyama _{1,

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Affiliation

(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 homology to other glycosyltransferases, and thus, significant ambiguity about its catalytic mechanism remains. 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 6550 ± 760 mer, consistent with the reported degree of polymerization of (1,3)-β-d-glucan isolated from intact cells. Pre-steady state kinetic analysis revealed a highly efficient but rate-determining chain elongation rate of 51.5 ± 9.8 s-1, which represents the first observation of chain elongation by a nucleotide-sugar-dependent polysaccharide synthase. Coupling the SEC-PAD-RC method with substrate analogue mechanistic probes provided the first unambiguous evidence that GS catalyzes non-reducing end polymerization. On the basis of 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.

中文翻译：

（1,3）-β-d-葡聚糖合酶在真菌细胞壁生物合成中的长度特异性和聚合机理

（1,3）-β-d-葡聚糖合酶（GS）催化真菌细胞壁中线性（1,3）-β-d-葡聚糖的形成，是临床认可的抗真菌抗生素的目标。GS的催化亚基FKS蛋白与其他糖基转移酶的序列同源性不高，因此，其催化机理仍然存在很大歧义。研究GS的主要技术障碍之一是缺少活性分析方法，该方法无法表征（1,3）-β-d-葡聚糖的长度和量，因为它在水和有机溶剂中的溶解性较差。在这里，我们报告了一种基于体积排阻色谱结合脉冲安培检测和辐射计数（SEC-PAD-RC）的新型GS活性测定方法的成功开发，这允许同时表征聚合物产物的量和长度。该测定法揭示了纯化的酵母GS产生的葡聚糖的长度为6550±760mer，这与从完整细胞分离的（1，3）-β-d-葡聚糖的报道的聚合度一致。稳态前动力学分析显示，高效但决定速率的链延长率为51.5±9.8 s-1，这是对核苷酸糖依赖性多糖合酶的链延长的首次观察。SEC-PAD-RC方法与底物类似物机械探针的耦合提供了第一个明确的证据，即GS催化非还原性末端聚合。基于这些观察，我们提出了GS催化机理的详细模型。

更新日期：2020-01-15

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