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糖胺聚糖。它与各种分子相互作用，并表现出重要的生物学功能。由大肠杆菌菌株K5产生的K5抗原是由GlcUA β 1 -4和GlcNAc α 1 -4重复二糖组成的线性多糖N-乙酰肝素原，其形成硫酸乙酰肝素的骨架。在这里，我们表达和纯化重组KfiA和KfiC蛋白，然后表征这些酶。尽管单独的重组KfiC没有表现出GlcUA转移酶活性，但它在KfiA存在下确实表现出GlcUA转移酶和聚合活性。相反，KfiA本身具有GlcNAc转移酶活性，其不受KfiC存在的影响。用KfiA和KfiC的各种截短突变体和点突变体分析GlcNAc和GlcUA转移酶活性。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体，以及删除C-末端和N-末端位点的截短突变体，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的相互作用是KfiC的GlcUA转移酶活性所必需的，但不是KfiA的酶活性所必需的。总之，这些结果表明KfiA和KfiC的复合物具有合成N-乙酰肝素原的聚合酶活性，为确定的硫酸乙酰肝素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.