Jing Wei, DeAngelis Paul L
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA.
Glycobiology. 2003 Oct;13(10):661-71. doi: 10.1093/glycob/cwg085. Epub 2003 Jun 10.
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.
A型多杀巴斯德氏菌产生透明质酸（HA）荚膜以增强感染能力。由972个残基组成的HA合酶pmHAS，可将由交替的β3-N-乙酰葡糖胺（GlcNAc）-β4-葡糖醛酸（GlcUA）构成的线性HA多糖聚合起来。我们之前已证明pmHAS拥有两个独立的糖基转移酶位点。在此我们进一步明确这些位点及推测的基序。缺失1至117位残基不影响HA聚合活性。GlcUA转移酶的羧基末端边界位于686至703位残基内。两个转移酶位点均含有对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，这进一步证实了我们对合酶多肽内两个活性位点的定位。利用高浓度UDP-糖进行的实验支持了DXD基序在底物结合中的潜在作用，这些实验部分挽救了某些突变体的活性。WGGED序列基序参与GlcNAc转移酶活性，因为在E³⁶⁹或D³⁷⁰处发生替换的突变体仅具有GlcUA转移酶活性。F型多杀巴斯德氏菌合成一种无硫酸化的软骨素（β3-GalNAc-β4-GlcUA）荚膜。一种由pmHAS的1至427位残基与同源软骨素合酶pmCS的421至704位残基组成的嵌合酶是一种有活性的HA合酶。由pmCS的1至420位残基与pmHAS的428至703位残基组成的反向嵌合酶是一种功能性软骨素合酶。对一组pmHAS/pmCS嵌合酶的分析确定了一个44个残基的区域（对应于pmHAS的225至265位残基），该区域参与UDP-己糖胺选择性。总体而言，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。
