Andreadeli A, Flemetakis E, Axarli I, Dimou M, Udvardi M K, Katinakis P, Labrou N E
Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece.
Biochim Biophys Acta. 2009 Jun;1794(6):976-84. doi: 10.1016/j.bbapap.2009.02.009. Epub 2009 Mar 10.
Formate dehydrogenases (FDHs, EC 1.2.1.2) comprise a group of enzymes found in both prokaryotes and eukaryotes that catalyse the oxidation of formate to CO(2). FDH1 from the model legume Lotus japonicus (LjFDH1) was cloned and expressed in E. coli BL21(DE3) as soluble active protein. The enzyme was purified using affinity chromatography on Cibacron blue 3GA-Sepharose. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of LjFDH1, based on the known structure of the Pseudomonas sp. 101 enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The temporal expression pattern of LjFDH1 gene was studied by real-time RT-PCR in various plant organs and during the development of nitrogen-fixing nodules. Furthermore, the spatial transcript accumulation during nodule development and in young seedpods was determined by in situ RNA-RNA hybridization. These results considered together indicate a possible role of formate oxidation by LjFDH1 in plant tissues characterized by relative hypoxia.
甲酸脱氢酶（FDHs，EC 1.2.1.2）是一类在原核生物和真核生物中均存在的酶，可催化甲酸氧化为二氧化碳。从模式豆科植物百脉根（Lotus japonicus）中克隆出FDH1（LjFDH1），并在大肠杆菌BL21(DE3)中表达为可溶性活性蛋白。该酶通过在Cibacron blue 3GA-琼脂糖凝胶上进行亲和层析进行纯化。研究了重组酶的酶学性质，并测定了多种底物的动力学参数（K(m)，k(cat)）。还基于假单胞菌属（Pseudomonas sp.）101酶的已知结构，通过分子建模研究构建了LjFDH1的模型。该分子模型用于帮助解释有关该酶底物特异性和催化机制的生化数据。通过实时RT-PCR研究了LjFDH1基因在各种植物器官以及固氮根瘤发育过程中的时间表达模式。此外，通过原位RNA-RNA杂交确定了根瘤发育过程中和幼嫩豆荚中的空间转录本积累。综合这些结果表明，LjFDH1催化的甲酸氧化在以相对缺氧为特征的植物组织中可能发挥作用。
