Batista F R, Hernández L, Fernández J R, Arrieta J, Menéndez C, Gómez R, Támbara Y, Pons T
Centre for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 10600, Cuba.
Biochem J. 1999 Feb 1;337 ( Pt 3)(Pt 3):503-6.
beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.
β-呋喃果糖苷酶具有一个保守的含天冬氨酸基序（精氨酸-天冬氨酸-脯氨酸；RDP），而α-吡喃葡萄糖苷酶没有该基序。通过定点诱变研究了重氮营养醋杆菌SRT4的果聚糖蔗糖酶（EC 2.4.1.10）的RDP基序中309位天冬氨酸的作用。用天冬酰胺取代309位天冬氨酸不影响酶的分泌。突变型果聚糖蔗糖酶的kcat降低了75倍，但其Km与野生型酶相似，表明309位天冬氨酸在催化中起主要作用。两种果聚糖蔗糖酶在pH 5.0时表现出最佳活性，并产生相似的产物谱。因此，D309N突变影响了蔗糖水解效率，但不影响酶的特异性。由于RDP基序在果糖基转移酶、转化酶、果聚糖酶、菊粉酶和蔗糖-6-磷酸水解酶中处于保守位置，它可能在β-呋喃果糖苷酶中具有共同的功能作用。
