Molecular analysis of two novel carbohydrate phosphorylases

瘤胃厌氧杆菌，白色瘤胃球菌，有两种甘露糖磷酸酶同工酶(类型-I和类型-II)，将甘露糖磷酸化为α-甘露糖1-磷酸和葡萄糖。底物专一性分析表明，类型-I对甘露糖具有专一性，但类型-II对甘露低聚糖的活性高于甘露糖。这些酶在合成反应中表现出不同的受体底物专一性：与对这些底物完全惰性的类型II酶不同，类型-I酶对6-羟基葡萄糖衍生物具有合成活性。通过序列比对和定点突变分析，确定I型酶的Asp129为催化氨基酸残基。在合成反应(逆反应)中，I型酶的Ile212对于识别6-羟基葡萄糖衍生物作为受体底物是重要的。

A ruminal anaerobic baceteium, Ruminococcus albus, has two mannosylglucose phosphorylase isozymes (Type-I and Type-II), phosphorolyzing mannosylglucose to alpha-mannose 1-phosphate and glucose. Anyalysis of substrate specificity revealed that Type-I was specific to mannosylglucose, but Type-II had higher activity to mannooligosaccharide than mannosylglucose. These enzymes showed different acceptor substrate specificity in the synthetic reaction: Type-I enzyme showed synthetic activity to 6-OH glucose derivatives unlike in contrast to Type-II, which was completely inert to these substrates. Asp129 of Type-I enzyme was determined to be catalytic amino acid residue based on sequence comparison and site-directed mutagenesis analysis. Ile212 of Type-I enzyme was important for recognition of 6-OH glucose derivatives as acceptor substrates in the synthetic reaction (reverse reaction).