Tsujimoto Yoshiyuki, Tanaka Hiroyuki, Takemura Reiko, Yokogawa Tomohiko, Shimonaka Atsushi, Matsui Hiroshi, Kashiwabara Shin-ichi, Watanabe Kunihiko, Suzuki Yuzuru
Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan.
J Biochem. 2007 Jul;142(1):87-93. doi: 10.1093/jb/mvm110. Epub 2007 May 24.
Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.
嗜热栖热芽孢杆菌α-1,4-葡萄糖苷酶（BS）对麦芽糖、麦芽寡糖和α-葡聚糖的α-1,4-糖苷键具有高度特异性。嗜热栖热芽孢杆菌寡-1,6-葡萄糖苷酶（BT）能特异性水解异麦芽糖、异麦芽寡糖和α-极限糊精的α-1,6键。这两种酶在一级结构上具有高度同源性，属于糖苷水解酶家族13，该家族包含四个保守区域（I、II、III和IV）。尽管这两种酶的底物特异性存在严格差异，但它们的结构被认为非常相似。通过定点诱变分析了这两种酶中底物识别的分子决定因素。构建并表征了20个基于BT的突变体和3个基于BS的突变体。与BS相比，BT在区域II中Val200→Ala和区域III中Pro258→Asn的双重取代导致了麦芽糖酶活性的出现，而异麦芽糖酶活性大幅降低。BT突变体对麦芽糖和异麦芽糖的k(0)/K(m)（s(-1). mM(-1)）值分别为69.0和15.4。我们得出结论，α-葡萄糖苷酶中的Val/Ala200和Pro/Asn258残基可能在很大程度上负责底物识别，尽管区域I和IV也有轻微影响。此外，BT V200A和V200A/P258N对蔗糖具有高水解活性。
