Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase:: two active sites exist in one polypeptide
Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase:: two active sites exist in one polypeptide
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DOI：
10.1093/glycob/10.9.883
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发表时间：
2000-09-01
期刊：
影响因子：
4.3
通讯作者：
DeAngelis, PL
中科院分区：
文献类型：
--
作者：
Jing, W;DeAngelis, PL
A型多杀性巴氏杆菌是一种动物病原体，采用透明质酸[HA]胶囊来避免宿主防御。PmHAS，972个残基的膜相关透明质酸合酶，催化GlcNAc和GlcUA两者的转移以形成HA聚合物。为了确定催化和膜相关结构域，分析了pmHAS突变体。PmHAS（1-703）是一种可溶的、有活性的HA合酶，表明羧基末端参与天然酶的膜结合，PmHAS（1-650）作为HA合酶是无活性的，但保留GlcNAc-转移酶活性。在pmNAS序列内，存在含有短基序Asp-Gly-Ser的重复结构域，其在许多β-糖基转移酶中是保守的。将这一天冬氨酸在任一结构域中改变为天冬酰胺、谷氨酸或赖氨酸将WA合酶活性降低至低水平。在残基196处取代的突变体具有GlcUA-转移酶活性，而在残基477处取代的突变体具有GlcNAc-转移酶活性。各种突变体的功能性转移酶活性的米氏常数，酶对前体的表观亲和力的量度，与野生型值相似。此外，在相同反应中混合D196 N和D477 K突变蛋白允许HA聚合在与野生型酶相似的水平。这些结果提供了合成酶多肽利用两个单独的糖基转移酶位点的第一个直接证据。
Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS(1-703) is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme, PmHAS(1-650) is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmNAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the WA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme, These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.