Combinatorial Engineering of Dextransucrase Specificity

We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.104 clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide 460DYVHT464 in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.本研究采用组合工程策略，探究了肠膜明串珠菌（Leuconostoc mesenteroides）NRRL B-512F来源的葡聚糖蔗糖酶DSR-S（dextransucrase DSR-S）的结构与糖苷键连接特异性之间的关联，并成功获得了连接特异性发生改变的酶突变体。通过对糖苷水解酶家族70（glycoside-hydrolase family 70）酶类的序列与结构分析，我们选定8个氨基酸位点（D306、F353、N404、W440、D460、H463、T464及S512）作为靶标，通过饱和诱变实现随机化并同步进行重组。对总计3.6×10^4个克隆的两个文库进行筛选后，我们分离得到包含81种突变体的工具库，这些突变体可合成分子量较高的α-葡聚糖（α-glucans），其α(1→3)糖苷键（α(1→3) linkages）占比范围为3%至20%。对突变体开展序列分析、生化表征及分子建模研究后，我们揭示了肽段460DYVHT464此前未被认知的功能：该肽段参与调控DSR-S的糖苷键连接特异性。该肽段序列与S512残基共同参与定义酶的+2亚位点拓扑结构（subsite topology），这一结构可能对该酶的区域特异性（regiospecificity）至关重要。