Tracy Breca S, Avci Fikri Y, Linhardt Robert J, DeAngelis Paul L
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2007 Jan 5;282(1):337-44. doi: 10.1074/jbc.M607569200. Epub 2006 Nov 10.
The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.
来自革兰氏阴性菌多杀性巴氏杆菌的透明质酸（HA）合酶PmHAS和硫酸软骨素合酶PmCS分别聚合糖胺聚糖（GAG）糖链HA或硫酸软骨素。先前已证明，重组大肠杆菌衍生的酶可延长其同源GAG的外源供应寡糖（例如，PmHAS延长HA）。在此我们表明，某些非同源GAG的寡糖和多糖（包括硫酸化和含艾杜糖醛酸的形式）可被PmHAS（例如，PmHAS延长硫酸软骨素）或PmCS延长。在合酶用单个单糖或长链（约10(2-4)个糖）延长分子的测定中测试了各种受体。与同源分子相比，某些GAG是非常差的受体，但仍检测到了延长产物。总体而言，这些发现表明，对于受体与酶之间的相互作用，（a）己糖胺C-4位羟基的取向并不关键，（b）己糖醛酸C-5的构象（葡萄糖醛酸与艾杜糖醛酸）并不重要，并且（c）在某些情况下，额外的负硫酸根基团具有良好的耐受性，例如在己糖胺的C-6位，但其他基团，包括C-4硫酸盐，则不被耐受或耐受性较差。在体内，细菌酶仅处理未硫酸化的聚合物；因此，预计PmCS和PmHAS催化剂不会通过作用于多种动物来源的硫酸化或差向异构化GAG而表现出这种相对宽松的糖特异性。然而，这一特性允许通过化学酶法合成多种嵌合GAG聚合物，包括蛋白聚糖复合物的模拟物。
