Sola-Carvajal Agustín, Gil-Ortiz Fernando, García-Carmona Francisco, Rubio Vicente, Sánchez-Ferrer Álvaro
*Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
†Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Valencia, Spain.
Biochem J. 2014 Sep 15;462(3):499-511. doi: 10.1042/BJ20140266.
NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+-dependent oxidation of ManNAc (N-acetyl-D-mannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH 9.4) with a high thermal stability in glycine buffer (Tm=64°C) and a strict selectivity towards ManNAc and NAD+. Crystal structures of ligand-free and ManNAc- and NAD+-bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences.
来自土壤拟杆菌黄杆菌属141 - 8的NAMDH（N - 乙酰 - D - 甘露糖胺脱氢酶）催化一种罕见的依赖NAD⁺的反应，将N - 乙酰 - D - 甘露糖胺（ManNAc）氧化为N - 乙酰甘露糖胺内酯，后者会自发水解为N - 乙酰甘露糖胺酸。NAMDH属于短链脱氢酶/还原酶（SDR）超家族，是迄今为止唯一被鉴定的NAMDH。为了更好地理解这种独特酶结构和生化方面的性质，已经进行了全面的功能、稳定性、定点诱变和晶体学研究。NAMDH在碱性pH（pH 9.4）下表现出显著的最佳活性，在甘氨酸缓冲液中具有高热稳定性（熔点Tm = 64°C），并且对ManNAc和NAD⁺具有严格的选择性。无配体、结合ManNAc和NAD⁺的酶形式的晶体结构显示，该酶为紧密的同四聚体，具有222点对称性，由呈现特征性SDR α3β7α3三明治折叠的亚基组成。用作连接相邻亚基的闩锁的高度发达的C末端尾巴稳定了四聚体。与底物的密集极性相互作用网络，包括其乙酰氨基基团包裹在特定结合口袋中以及糖4 - OH原子的氢键结合，确保了对ManNAc的特异性。NAMDH - 底物复合物和定点诱变研究确定了催化四联体，并为鉴定新的NAMDH序列提供了有用的特征。
