Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Enzyme Microb Technol. 2011 Jun 10;49(1):59-65. doi: 10.1016/j.enzmictec.2011.03.010. Epub 2011 Apr 7.
Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from l-rhamnose to GlcNAc, GalNAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc. These results confirm the correlation between the amino acid residue at position 162 and the enzyme's specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GalNAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GlcNAc as acceptor, which illustrates that the structure-function relationships in β-galactoside phosphorylases are not yet completely understood. Several other positions have also been examined by mutational analysis but true determinants of the acceptor specificity in family GH-112 could not be identified.
糖苷磷酸化酶是一类特殊的碳水化合物活性酶，具有糖苷水解酶和糖基转移酶之间的特性。来自 GH-112 家族的磷酸化酶是例外的，因为它们使用半乳糖-1-磷酸而不是葡萄糖-1-磷酸作为糖基供体。在这个家族中观察到了不同的受体特异性，范围从 l-鼠李糖到 GlcNAc、GalNAc 以及后者的组合。现在已经对来自 GH-112 家族进化枝中以前未探索的分支的三种新的磷酸化酶进行了特征描述，以更深入地了解这种受体特异性的差异。来自猪红斑丹毒丝菌和念珠状链杆菌的酶被发现更喜欢 GalNAc 作为受体，而来自厌氧球菌的酶在 GalNAc 和 GlcNAc 上显示出相似的活性。这些结果证实了位置 162 上的氨基酸残基与酶特异性之间的相关性，即前者组中的苏氨酸和后者组中的缬氨酸。然而，残基 162 的诱变不能合理地改变底物偏好，因为双歧杆菌中酶的缬氨酸被苏氨酸取代并没有使其对 GalNAc 的特异性更严格。出乎意料的是，在位置 162 引入异亮氨酸增加了对 GlcNAc 作为受体的偏好，这表明β-半乳糖苷磷酸化酶的结构-功能关系尚未完全理解。通过突变分析还检查了其他几个位置，但在 GH-112 家族中无法确定受体特异性的真正决定因素。
