Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus.
Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus.
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DOI：
10.1042/bj20100351
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发表时间：
2010-10
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通讯作者：
E. Yoshida;Masafumi Hidaka;S. Fushinobu;T. Koyanagi;Hiromichi Minami;H. Tamaki;M. Kitaoka;T. Katayama;H. Kumagai
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作者：
E. Yoshida;Masafumi Hidaka;S. Fushinobu;T. Koyanagi;Hiromichi Minami;H. Tamaki;M. Kitaoka;T. Katayama;H. Kumagai
β糖苷酶(KmBglI)属于糖苷水解酶家族3。该酶的特别不同寻常之处在于，PA14结构域(Pf07691)被插入到催化核心序列中，该结构域已被声称具有碳水化合物结合作用。在本研究中，我们测定了KmBglI在2.55A(1 A=0.1 nm)分辨率下与葡萄糖形成的络合物的酶性质和晶体结构。KmBglI的一个显著特点是酶的活性基本上局限于二糖，当以三糖为底物时，酶的活性急剧下降。这种链长特异性与大麦β-D-葡聚糖水解酶(ExoI)对寡糖的首选作用形成鲜明对比，ExoI没有PA14结构域插入。KmBglI的(-1)亚基的结构与耐热菌β-葡萄糖苷酶的结构基本相同，也与ExoI相似，但(+1)亚基的结构在它们之间有很大的不同。在KmBglI中，从PA14结构域延伸出来的环覆盖在催化口袋上形成亚位(+1)，因此同时成为空间位阻，可能限制底物的链长。突变研究证实了环区在决定底物特异性中的关键作用。KmBglI的PA14结构域介导的活性位点的形成调用了其N-末端结构域对α-半乳糖苷酶的β互补作用，而PA14结构域与其结构相似。本研究首次揭示了PA14结构域与碳水化合物相互作用的结构基础。
β-Glucosidase from Kluyveromyces marxianus (KmBglI) belongs to the GH3 (glycoside hydrolase family 3). The enzyme is particularly unusual in that a PA14 domain (pf07691), for which a carbohydrate-binding role has been claimed, is inserted into the catalytic core sequence. In the present study, we determined the enzymatic properties and crystal structure of KmBglI in complex with glucose at a 2.55 A (1 A=0.1 nm) resolution. A striking characteristic of KmBglI was that the enzyme activity is essentially limited to disaccharides, and when trisaccharides were used as the substrates the activity was drastically decreased. This chain-length specificity is in sharp contrast with the preferred action on oligosaccharides of barley β-D-glucan glucohydrolase (ExoI), which does not have a PA14 domain insertion. The structure of subsite (-1) of KmBglI is almost identical with that of Thermotoga neapolitana β-glucosidase and is also similar to that of ExoI, however, the structures of subsite (+1) significantly differ among them. In KmBglI, the loops extending from the PA14 domain cover the catalytic pocket to form subsite (+1), and hence simultaneously become a steric hindrance that could limit the chain length of the substrates to be accommodated. Mutational studies demonstrated the critical role of the loop regions in determining the substrate specificity. The active-site formation mediated by the PA14 domain of KmBglI invokes α-complementation of β-galactosidase exerted by its N-terminal domain, to which the PA14 domain shows structural resemblance. The present study is the first which reveals the structural basis of the interaction between the PA14 domain and a carbohydrate.