PURIFICATION AND PROPERTIES OF CLONED SALMONELLA-TYPHIMURIUM LT2 SIALIDASE WITH VIRUS-TYPICAL KINETIC PREFERENCE FOR SIALYL ALPHA-2-]3 LINKAGES
PURIFICATION AND PROPERTIES OF CLONED SALMONELLA-TYPHIMURIUM LT2 SIALIDASE WITH VIRUS-TYPICAL KINETIC PREFERENCE FOR SIALYL ALPHA-2-]3 LINKAGES
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DOI：
10.1093/oxfordjournals.jbchem.a123603
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发表时间：
1991-09-01
影响因子：
2.7
通讯作者：
VIMR, ER
中科院分区：
文献类型：
--
作者：
HOYER, LL;ROGGENTIN, P;VIMR, ER
含有鼠伤寒沙门氏菌LT2唾液酸酶基因nanH的亚克隆在大肠杆菌中从pBR329的多拷贝衍生物表达。所克隆的唾液酸酶结构基因指导唾液酸酶多肽的过量产生，所述唾液酸酶多肽被检测为无细胞提取物中的主要可溶性蛋白种类。过量生产的酶纯化后，使用常规的制备技术的65倍富集电泳均匀性。与以前研究的所有唾液酸酶不同，S.鼠伤寒唾液酸酶带正电荷（pI大于或等于9.0）。使用2'-（4-甲基伞形酮基）-α-D-N-乙酰神经氨酸（MUNeu5Ac）测定的纯化唾液酸酶的K（m）、V（max）和转换数分别为0.25 mM、5，200 nmol min-1和2，700 s-1。这些值是唾液酸酶报告的最高值。唾液酸酶被2-脱氧-2，3-二脱氢-N-乙酰神经氨酸在异常高的浓度下抑制（K（i）= 0.38 mM），但不被20 mM N-乙酰神经氨酸抑制。活性不需要二价阳离子。MUNeu5Ac水解的最适pH值为5.5 - 7.0，取决于测定缓冲液系统。使用天然唾液酸糖缀合物的底物特异性测量显示，与α-2-> 6结合的唾液酸相比，唾液酸α-2-> 3键的动力学偏好性为260倍。该酶还有效地切割糖蛋白和神经节苷脂的残基，但不能切割粘蛋白或唾液酸同多糖。S.因此，鼠伤寒唾液酸酶是第一种被描述为具有甲型流感病毒唾液酸酶样对唾液酸α-2-> 3键的动力学偏好并具有碱性pI的细菌酶。
Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater-than-or-equal-to 9.0). K(m), V(max), and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (K(i) = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha-2 --> 3 linkages when compared with alpha-2 --> 6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha-2 --> 3 linkages and to have a basic pI.