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Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ( IF 3.2 ) Pub Date : 2020-06-26 , DOI: 10.1016/j.bbapap.2020.140476
Hironaga Akita 1 , Yusuke Nakamichi 1 , Tomotake Morita 2 , Akinori Matsushika 1
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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.



中文翻译:

脂解嗜热嗜热丝菌NAD(P)+依赖的介观二氨基己二酸酯脱氢酶的表征。

内消旋-Diaminopimelate脱氢酶(内消旋-DAPDH)催化可逆NADP + -依赖性氧化脱氨的内消旋-二氨基庚二酸-2,6-(内消旋-DAP),以产生-2-氨基-6- oxopimelate。根据底物特异性和结构特征,内消旋-DAPDH分为I和II型两个主要簇。在这里,我们描述了来自解脂菌(T1DAPDH)的新型II型内消旋-DAPDH 。编码推定的T1DAPDH的基因在大肠杆菌中表达细胞,然后从粗细胞提取物中将酶纯化7.3倍至同质。TlDAPDH分子似乎形成了六聚体,这是II型内消旋-DAPDHs的典型结构特征。纯化的酶以NADP +和NAD +为辅酶,对内消旋-DAP表现出氧化脱氨活性。T1DAPDH表现出相应的2-氧代酸的还原胺化活性以产生d-氨基酸。特别地,在II型酶中没有报道d-天冬氨酸和d-谷氨酸的生产率。中氧化脱氨的最佳pH和温度-DAP分别为10.5和55℃。在50°C至65°C的温度以及4.5-9.5的pH范围内孵育30分钟后,TlDAPDH保留了其80%以上的活性。此外,基于多序列比对和同源性模型阐明了T1DAPDH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理与嗜热链球菌的中-DAPDH相似,后者是典型的II型酶。基于动力学特征和结构比较,认为T1DAPDH是新型的II型内消旋-DAPDH。

更新日期:2020-07-10
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