Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.
Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.
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DOI：
10.1007/s00253-013-4947-1
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发表时间：
2014-03
影响因子：
5
通讯作者：
Chen, Xi
中科院分区：
文献类型：
--
作者：
Li, Yanhong;Yu, Hai;Thon, Vireak;Chen, Yi;Muthana, Musleh M.;Qu, Jingyao;Hie, Liana;Chen, Xi
关键词：
硫酸乙酰肝素（HS）和肝素（HP）的生物活性与其分子结构密切相关。多杀性巴氏杆菌肝素原合酶2（PmHS 2）和大肠杆菌K5 KfIA两者已用于HS和HP寡糖及其衍生物的酶促和化学酶促合成。我们在这里表明，使用pET 15 b载体克隆并表达PmHS 2作为N-His 6-taged融合蛋白提高了其在E.杆菌对多杀性巴氏杆菌肝素原合酶2（PmHS 2）和大肠杆菌K5 KfiA的N-乙酰葡糖胺基转移酶活性的供体底物特异性的研究表明PmHS 2和KfiA的底物混杂性。总之，PmHS 2和KfiA都可以使用尿苷5 '-二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）及其一些C2'-和C6 '-衍生物作为其α1-4-GlcNAcT活性的供体底物。然而，PmHS 2对底物修饰具有更宽的耐受性。除了可被KfiA使用的UDP-糖之外，UDP-GlcNAc、UDP-葡萄糖和UDP-N-乙酰半乳糖胺（UDP-GalNAc）的其他C6 '-衍生物是PmHS 2的α1-4-GlcNAcT活性的可耐受底物。PmHS 2和KfIA的底物混杂性将允许有效的化学酶促合成不同的HS和HP寡糖衍生物，其与其天然对应物相比可能具有改善或改变的活性。
The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2’- and C6’-derivatives as donor substrates for their α1–4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6’-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1–4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.
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DOI：
10.1126/science.1207478
发表时间：
2011-10-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Xu Y;Masuko S;Takieddin M;Xu H;Liu R;Jing J;Mousa SA;Linhardt RJ;Liu J
通讯作者：
Liu J
DOI：
10.1016/b978-0-12-396527-1.00003-6
发表时间：
2012
影响因子：
--
作者：
Dulaney, Steven B.;Huang, Xuefei
通讯作者：
Huang, Xuefei
影响因子：
4.8
作者：
Chavaroche, Anais A. E.;van den Broek, Lambertus A. M.;Eggink, Gerrit
通讯作者：
Eggink, Gerrit
影响因子：
3
作者：
Lindahl, U
通讯作者：
Lindahl, U
影响因子：
4.9
作者：
Chen, Yi;Thon, Vireak;Chen, Xi
通讯作者：
Chen, Xi