Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32, Kagamiyama, HigashiHiroshima, Hiroshima 739-0046, Japan.
J Struct Biol. 2019 Jan 1;205(1):84-90. doi: 10.1016/j.jsb.2018.11.005. Epub 2018 Nov 13.
Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1 → 6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α) TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite -1' played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites -1 and -1', respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.
产异头麦芽糖酶（IPases）从木葡聚糖寡糖的非还原端释放异头麦芽糖（α-d-吡喃木糖基-(1 → 6)-d-吡喃葡萄糖）。米曲霉 IPase（IpeA）被归类为糖苷水解酶家族 3（GH3）的成员；然而，与其他 GH3 酶相比，它具有不寻常的底物特异性。木葡聚糖寡糖非还原端的吡喃木糖支化对于 IpeA 活性至关重要。我们以 2.4 Å 的分辨率解析了与异头麦芽糖结合的 IpeA 的晶体结构，表明 IpeA 的结构形成二聚体，由三个结构域组成：N 端（β/α）TIM-桶结构域、α/β/α 夹心折叠结构域和 C 端纤维连接蛋白样结构域。催化 TIM-桶结构域具有催化亲核试剂（Asp300）和酸碱（Glu524）残基。有趣的是，我们发现 IpeA 的活性位点腔大于其他 GH3 酶，并且 -1'亚位点在其活性中起着重要作用。异头麦芽糖的吡喃葡萄糖基和吡喃木糖基残基分别位于 -1 和 -1'亚位点。Gln58 和 Tyr89 通过氢键和堆积作用分别与异头麦芽糖的吡喃木糖基残基相互作用。我们的发现为 GH3 酶的底物识别提供了新的见解。
