Matuschek M, Burchhardt G, Sahm K, Bahl H
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Federal Republic of Germany.
J Bacteriol. 1994 Jun;176(11):3295-302. doi: 10.1128/jb.176.11.3295-3302.1994.
The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.
来自嗜热栖热硫化叶菌EM1的支链淀粉酶基因（amyB）在大肠杆菌中克隆，并测定了核苷酸序列。amyB的阅读框由5586个碱基对组成，编码一种分子量为205991道尔顿的超大酶。序列分析揭示了支链淀粉酶由催化结构域和非催化结构域组成的复合结构。该蛋白的N端一半包含一个35个氨基酸残基的前导肽和催化结构域，其中包括淀粉酶的四个共有区域。几种支链淀粉酶共有区域的比较表明，像嗜热栖热硫化叶菌EM1的II型支链淀粉酶那样水解α-1,4-和α-1,6-糖苷键的酶在共有区域具有特定的氨基酸序列。这些与仅切割α-1,6键的I型支链淀粉酶不同。对酶功能不必要的C端一半至少由两个不同的片段组成。一个约70 kDa的片段包含两个III型纤连蛋白样结构域拷贝，后面是一个富含甘氨酸、丝氨酸和苏氨酸残基的连接区。在C端，我们发现了三个约50个氨基酸的重复序列，这些序列也存在于例如嗜热栖热菌和基伍产乙酸菌的表层（S层）蛋白的N端。由于已知嗜热栖热硫化叶菌EM1的支链淀粉酶是细胞结合型的，我们的结果表明该片段作为S层锚定物，使支链淀粉酶附着于细胞表面。因此，提出了一个细胞外酶附着于细胞表面的通用模型，该模型赋予S层一个新功能，并且可能在具有S层的细菌中广泛存在。这种三倍重复的S层样片段存在于不同细菌的几种酶中。在amyB上游，鉴定出另一个开放阅读框，编码一种35.6 kDa的假定蛋白。未发现与DNA和蛋白质数据库中其他可用序列有明显相似性。
