Ortiz-Soto Maria Elena, Rivera Manuel, Rudiño-Piñera Enrique, Olvera Clarita, López-Munguía Agustin
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos 62210, Mexico.
Protein Eng Des Sel. 2008 Oct;21(10):589-95. doi: 10.1093/protein/gzn036. Epub 2008 Jul 1.
Levansucrases (LS) are fructosyltransferases (FTFs) belonging to family 68 of glycoside hydrolases (GH68) using sucrose as substrate to synthesize levan, a fructose polymer. From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated. A product specificity modification was obtained with mutants Y429N and R433A that no longer produce levan but exclusively oligosaccharides. An effect of the mutation S164A was observed on enzyme stability and kinetic behavior; this mutation also induces a levan activation effect that enhances the reaction rate. We report the crystallographic structure of this mutant and found that S164 is an important residue to maintain the nucleophile position in the active site. We also found evidence of the important role of Y429 in acceptor specificity: this is a key residue coordinating the sucrose position in the catalytic domain-binding pocket. Some of these mutations resulted in LS with a broad range of specificities and new biochemical properties.
果聚糖蔗糖酶（LS）是属于糖苷水解酶68家族（GH68）的果糖基转移酶（FTF），它以蔗糖为底物合成果聚糖（一种果糖聚合物）。通过对GH68家族蛋白质的多序列分析，选择了9个残基，并研究了它们在受体和产物特异性以及枯草芽孢杆菌LS生化特性中的作用。突变体Y429N和R433A不再产生果聚糖，而是只产生寡糖，实现了产物特异性的改变。观察到突变S164A对酶稳定性和动力学行为有影响；该突变还诱导了果聚糖激活效应，提高了反应速率。我们报道了该突变体的晶体结构，发现S164是维持活性位点亲核试剂位置的重要残基。我们还发现了Y429在受体特异性中起重要作用的证据：这是一个在催化结构域结合口袋中协调蔗糖位置的关键残基。其中一些突变产生了具有广泛特异性和新生化特性的LS。
