Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
Biochim Biophys Acta Proteins Proteom. 2020 Oct;1868(10):140476. doi: 10.1016/j.bbapap.2020.140476. Epub 2020 Jun 26.
meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP and NAD as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5-9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.
甲二羟苯丙氨酸脱氢酶（meso-DAPDH）催化可逆的 NADP 依赖性氧化脱氨甲二羟苯丙氨酸（meso-DAP）生成 l-2-氨基-6-氧代庚二酸。meso-DAPDH 根据底物特异性和结构特征分为两个主要簇，I 型和 II 型。在这里，我们描述了来自Thermosyntropha lipolytica 的一种新型 II 型 meso-DAPDH（TlDAPDH）。编码 TlDAPDH 的基因在大肠杆菌细胞中表达，然后从粗细胞提取物中纯化为 7.3 倍的均相。TlDAPDH 的分子似乎形成六聚体，这是 II 型 meso-DAPDH 的典型结构特征。纯化的酶对 meso-DAP 表现出氧化脱氨活性，NADP 和 NAD 均可作为辅酶。TlDAPDH 对相应的 2-氧代酸具有还原胺化活性，可产生 d-氨基酸。特别是，在 II 型酶中尚未报道 d-天冬氨酸和 d-谷氨酸的产物。meso-DAP 氧化脱氨的最适 pH 和温度分别为 10.5 和 55°C。TlDAPDH 在 50°C 至 65°C 之间的温度下孵育 30 分钟后，在 pH 值为 4.5-9.5 的范围内保留超过 80%的活性。此外，基于多序列比对和同源模型阐明了 TlDAPDH 的辅酶和底物识别机制。这些分析的结果表明，TlDAPDH 的辅酶和底物识别的分子机制与作为代表性 II 型酶的来自 S. thermophilum 的 meso-DAPDH 相似。基于动力学特征和结构比较，TlDAPDH 被认为是一种新型的 II 型 meso-DAPDH。
