Chen Tai-Hung, Huang Yu-Chiao, Yang Chii-Shen, Yang Chien-Chih, Wang Ai-Yu, Sung Hsien-Yi
Institute of Microbiology and Biochemistry and Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
Phytochemistry. 2009 Jan;70(1):25-31. doi: 10.1016/j.phytochem.2008.10.004. Epub 2008 Nov 17.
Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobetafruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K(m) values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k(cat) values than the wild-type enzyme, but an increase in the k(cat) value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.
植物酸性转化酶与细胞壁相关或存在于液泡中，属于糖苷水解酶家族32（GH32）。以拟南芥细胞壁转化酶AtcwINV1为模板对竹子液泡转化酶Bobetafruct3进行同源建模，结果表明其整体结构与GH32酶相似，且三个高度保守的基序NDPNG、RDP和EC位于活性位点。本研究还利用定点诱变来研究这三个基序中保守氨基酸残基的作用，这些残基包括Asp135、Arg259、Asp260、Glu316和Cys317，以及位于NDPNG和RDP基序之间的一个保守色氨酸残基（Trp159）。突变体W159F、W159L、E316Q和C317A保留了酸性转化酶活性，但突变体E316A或Asp135、Arg259或Asp260发生变化的突变体未观察到转化酶活性。具有转化酶活性的四个突变体的表观K(m)值均高于野生型酶。突变体W159L和E316Q的k(cat)值低于野生型酶，但突变体W159F和C317A的k(cat)值有所增加。本研究结果表明，这些残基在催化蔗糖水解中具有各自的功能。
