Study on glucuronyltransferase activity of KfiA

硫酸乙酰肝素是大多数动物细胞外基质中普遍存在的一种糖胺多糖。它与多种分子相互作用，具有重要的生物学功能。由大肠杆菌K5菌株产生的K5抗原是由GlcUAβ1-4和GlcNAcα1-4重复的二糖组成的线性多糖N-乙酰肝素，构成了硫酸乙酰肝素的骨架。在此，我们表达和纯化了重组的KfiA和KfiC蛋白，并对这些酶进行了鉴定。单独的重组KfiC不显示GlcUA转移酶活性，但在KfiA存在的情况下，它确实具有GlcUA转移酶和聚合活性。相反，KfiA本身具有GlcNAc转移酶活性，不受KfiC存在的影响。用不同的KfiA和KfiC截短突变体和点突变株分析了GlcNAc和GlcUA转移酶的活性。取代DXD基序的天冬氨酸和离子氨基酸簇的赖氨酸和谷氨酸的点突变，以及缺失C-末端和N-末端的截短突变，分别揭示了KfiC和KfiA的GlcNAc和GlcUA转移酶活性的必需区域。KfiC与KfiA的相互作用对KfiC的GlcUA转移酶活性是必需的，但对KfiA的酶活性不是必需的。综上所述，这些结果表明KfiA和KfiC的络合物具有合成N-乙酰肝素的聚合酶活性，为明确的乙酰肝素硫酸酯链的生物工程提供了有用的工具。

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA beta1-4 and GlcNAc alpha1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.