Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute-GBB, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Appl Environ Microbiol. 2011 Nov;77(22):8154-63. doi: 10.1128/AEM.05735-11. Epub 2011 Sep 23.
Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (β/α)₈ barrel that use sucrose to synthesize α-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.
罗伊氏乳杆菌 121 使用葡糖基转移酶 A（GTFA）酶将蔗糖转化为大量的 α-D-葡聚糖 reuteran，一种胞外多糖。gtfA 上游还有另一个假定的葡聚糖蔗糖酶基因，命名为 gtfB。之前，我们已经表明，纯化的重组 GTFB 蛋白/酶与蔗糖不起作用。gtfB 的各种同源物存在于其他乳杆菌菌株中，包括 L. reuteri 模式菌株 DSM 20016，其基因组序列是可用的。在这里，我们报告 GTFB 是一种新型的 α-葡聚糖转移酶，具有歧化（裂解α1→4 并合成α1→6 和α1→4 糖苷键）和α1→6 聚合类型的活性，对麦芽四糖和更大的麦芽寡糖底物（简而言之，它是一种 4,6-α-葡聚糖转移酶）。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）、甲基化分析和一维 ¹H 核磁共振（NMR）光谱对麦芽七糖合成的化合物类型进行表征，发现仅生成线性产物，随着聚合度（DP）的增加，最终产物中引入更多的α1→6 糖苷键，从孵育混合物中的 18%到富集馏分中的 33%不等。鉴于其一级结构，GTFB 显然是糖苷水解酶 70（GH70）家族的成员，该家族包含具有置换（β/α）₈ 桶的酶，使用蔗糖合成α-D-葡聚糖聚合物。然而，GTFB 酶的反应和产物特异性对于 GH70 家族来说是新颖的，类似于使用麦芽寡糖作为底物的 GH13 α-淀粉酶型酶的特性，但在将一系列α1→6 糖苷键引入线性寡糖产物方面有所不同。我们得出结论，GTFB 代表 GH13 和 GH70 酶家族之间的一种新型进化中间体，我们推测了它的起源。
