Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima, Japan.
Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Microbiologyopen. 2020 Aug;9(8):e1059. doi: 10.1002/mbo3.1059. Epub 2020 Jun 2.
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. Moreover, d-amino acid dehydrogenase (d-AADHs) derived from protein-engineered meso-DAPDH is useful for one-step synthesis of d-amino acids with high optical purity. Here, we report the identification and functional characterization of a novel NAD(P) -dependent meso-DAPDH from Numidum massiliense (NmDAPDH). After the gene encoding the putative NmDAPDH was expressed in recombinant Escherichia coli cells, the enzyme was purified 4.0-fold to homogeneity from the crude extract through five purification steps. Although the previously known meso-DAPDHs use only NADP as a coenzyme, NmDAPDH was able to use both NADP and NAD as coenzymes. When NADP was used as a coenzyme, NmDAPDH exhibited an approximately 2 times higher k /K value toward meso-DAP than that of meso-DAPDH from Symbiobacterium thermophilum (StDAPDH). NmDAPDH also catalyzed the reductive amination of corresponding 2-oxo acids to produce acidic d-amino acids such as d-aspartate and d-glutamate. The optimum pH and temperature for the oxidative deamination of meso-DAP were about 10.5 and 75°C, respectively. Like StDAPDH, NmDAPDH exhibited high stability: it retained more than 75% of its activity after 30 min at 60°C (pH 7.2) or at pHs ranging from 5.5 to 13.0 (50°C). Alignment of the amino acid sequences of NmDAPDH and the known meso-DAPDHs suggested NmDAPDH has a hexameric structure. Given its specificity for both NADP and NAD , high stability, and a broad range of reductive amination activity toward 2-oxo acids, NmDAPDH appears to offer advantages for engineering a more effective d-AADH.
内消旋二氨基庚二酸脱氢酶（meso-DAPDH）催化可逆的 NADP 依赖性氧化脱氨反应，将内消旋-2,6-二氨基庚二酸（meso-DAP）转化为 l-2-氨基-6-氧代庚二酸。此外，通过蛋白质工程化的 meso-DAPDH 衍生的 d-氨基酸脱氢酶（d-AADH）可用于一步法合成具有高光学纯度的 d-氨基酸。在这里，我们报告了从 Numidum massiliense（NmDAPDH）中鉴定和功能表征的一种新型 NAD（P）依赖性 meso-DAPDH。在重组大肠杆菌细胞中表达编码假定的 NmDAPDH 的基因后，该酶通过五个纯化步骤从粗提物中以 4.0 倍的纯度进行了纯化。尽管以前已知的 meso-DAPDH 仅使用 NADP 作为辅酶，但 NmDAPDH 能够同时使用 NADP 和 NAD 作为辅酶。当使用 NADP 作为辅酶时，NmDAPDH 对 meso-DAP 的 k / K 值约为嗜热共生菌（StDAPDH）来源的 meso-DAPDH 的 2 倍。NmDAPDH 还催化相应的 2-酮酸的还原胺化反应，生成酸性 d-氨基酸，如 d-天冬氨酸和 d-谷氨酸。meso-DAP 氧化脱氨的最适 pH 和温度分别约为 10.5 和 75°C。与 StDAPDH 一样，NmDAPDH 表现出很高的稳定性：在 60°C（pH 7.2）或 pH 5.5 至 13.0（50°C）的条件下，30 分钟后保留了超过 75%的活性。NmDAPDH 与已知的 meso-DAPDH 的氨基酸序列比对表明，NmDAPDH 具有六聚体结构。鉴于其对 NADP 和 NAD 的特异性、高稳定性以及对 2-酮酸的广泛还原胺化活性，NmDAPDH 似乎为工程化更有效的 d-AADH 提供了优势。
