Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida
Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida
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DOI：
10.1093/glycob/cwg085
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发表时间：
2003-10-01
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型多杀性巴氏杆菌产生透明质酸（HA）胶囊以增强感染。972位残基HA合成酶pmHAS可聚合由β - 3n -乙酰氨基葡萄糖(GlcNAc)- β - 4 -葡萄糖醛酸（GlcUA）交替组成的线性HA多糖。我们先前证明pmHAS具有两个独立的糖基转移酶位点。在这里，我们进一步定义了地点和假定的母题。残基1-117的缺失不影响HA聚合活性。glcua转移酶的羧基端边界位于残基686-703内。两个转移酶位点都包含一个对HA合酶活性至关重要的DXD基序。D247N或D249N突变体仅具有glcua转移酶活性，而D527N或D529N突变体仅具有glcnac转移酶活性，进一步证实了我们在合成酶多肽中分配的两个活性位点。利用高udp -糖浓度的实验支持了DXD基序在底物结合中的潜在作用，该实验部分地挽救了某些突变体的活性。WGGED序列基序与glcnac转移酶活性有关，因为替换E369或D370的突变体仅具有glcua转移酶活性。多毒型F型合成了一种无硫酸软骨素（β - 3galnac - β - 4glcua）胶囊。由pmHAS残基1 ~ 427和pmCS残基421 ~ 704组成的嵌合酶是一种具有活性的HA合成酶。由pmCS残基1 ~ 420和pmHAS残基428 ~ 703组成的反向嵌合酶是功能性的软骨素合成酶。对pmHAS/pmCS嵌合酶的分析发现了一个44个残基区域，对应pmHAS残基225-265，参与了udp -己糖胺的选择性。总的来说，这些发现进一步支持了单个多肽内两个独立转移酶位点的模型。
Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.