Meissner H, Liebl W
Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.
Eur J Biochem. 1998 Dec 15;258(3):1050-8. doi: 10.1046/j.1432-1327.1998.2581050.x.
A novel enzyme acting on starch and malto-oligosaccharides was identified and characterised. The non-hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto-oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents. i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto-oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X +/- 2n (with X > or = 3 and n = 0,1,2,...). Maximum activity in a 10-min assay was recorded at pH 6.5 and 85-90 degrees C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70 degrees C (pH 6.5, 0.34 mg ml-1 protein), MTase half-lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded. The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low-level local similarity between MTase and the alpha-amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (beta/alpha)8-fold as the core supersecondary structure which is typical for the alpha-amylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the alpha-amylase family, inserted between beta-strand 3 and alpha-helix 3 of the (beta/alpha)8-barrel fold).
一种作用于淀粉和麦芽寡糖的新型酶被鉴定并表征。嗜热栖热菌（Thermotoga maritima）MSB8的这种非水解酶被命名为麦芽寡糖基转移酶（MTase），它通过糖基转移反应使麦芽寡糖发生歧化反应。该酶具有独特的转移特异性，严格局限于麦芽寡糖基单元的转移。将MTase与淀粉或其成分（即直链淀粉和支链淀粉）一起温育，会导致形成一系列麦芽糖倍数的产物（即麦芽糖、麦芽四糖、麦芽六糖等）。聚合度（DP）为X的麦芽寡糖会歧化为DP为X±2n的产物（其中X≥3且n = 0、1、2……）。在10分钟的测定中，pH 6.5和85 - 90℃时记录到最大活性。该酶对热失活表现出非凡的抗性。例如，在90℃、85℃和70℃（pH 6.5，蛋白质浓度0.34 mg/ml）时，MTase的半衰期分别约为2.5小时、17小时和21天。从嗜热栖热菌MSB8菌株的基因文库中分离出了MTase基因，命名为mmtA。根据mmtA核苷酸序列推导的MTase一级结构分析表明，该酶与已知蛋白质序列关系不密切。然而，检测到MTase与α -淀粉酶酶家族（糖基水解酶家族13）之间存在低水平的局部相似性，包括催化所必需的保守酸性残基。因此，MTase应归属于该家族。基于详细的序列分析以及与已知晶体结构的淀粉分解酶的比较，我们提出MTase含有一个（β/α）8折叠作为核心超二级结构，这是α -淀粉酶家族的典型结构。另一方面，MTase的独特之处在于它缺少该家族中高度保守的几个残基。此外，MTase拥有一个异常大的结构域B（α -淀粉酶家族的典型结构域，插入在（β/α）8桶状折叠的β链3和α螺旋之间）。
