Analysis of the Polymerization Initiation and Activity of Pasteurella multocida Heparosan Synthase PmHS2, an Enzyme with Glycosyltransferase and UDP-sugar Hydrolase Activity
Analysis of the Polymerization Initiation and Activity of Pasteurella multocida Heparosan Synthase PmHS2, an Enzyme with Glycosyltransferase and UDP-sugar Hydrolase Activity
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DOI：
10.1074/jbc.m110.136754
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发表时间：
2011-01-21
影响因子：
4.8
通讯作者：
Eggink, Gerrit
中科院分区：
文献类型：
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作者：
Chavaroche, Anais A. E.;van den Broek, Lambertus A. M.;Eggink, Gerrit
乙酰肝素合成酶通过交替地将UDP-GlcUA和UDP-GlcNAc的单糖单元转移到受体分子上，催化肝素(-4GlcUAβ1-4GlcNAcα1-)(N)的聚合。巴氏杆菌多杀性葡聚糖合成酶PmHS2引发葡聚糖链及其对聚合过程的影响尚未见报道。通过对PmHS2进行定点突变，获得了PmHS2-GlcUA(+)和PmHS2-GlcNAc(+)的单作用转移酶。当在标准聚合条件下孵育时，PmHS2-GlcUA(+)/PmHS2-GlcNAc(+)具有与PmHS2相似的聚合性能。我们利用单作用转移酶，通过研究PmHS2在肝素模板和不同UDP-糖浓度存在下的聚合过程，研究了肝素链引发的第一步。我们观察到，当PmHS2在过量的UDP-GlcNAc存在的情况下孵育时，有利于肝素链的启动。这导致了较多的平均相对分子质量较低的肝素链，或者分别在没有或存在肝素模板的情况下合成了两组不同长度的肝素糖链。这些数据表明，PmHS2将GlcUA从UDP-GlcUA部分转移到UDP-GlcNAc受体分子上，从而引发了葡聚糖聚合；因此，不仅UDP-糖的浓度，而且每个UDP-糖的量都影响着PmHS2的聚合过程。此外，PmHS2还能降解UDP-糖，其中UDP-GlcUA的降解率高于UDP-GlcNAc。然而，在两种UDP-糖存在下孵育的PmHS2比UDP-糖更有利于合成肝素聚合物而不是UDP-糖。
Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUA beta 1-4GlcNAc alpha 1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.