Cloning and characterization of Lotus japonicus formate dehydrogenase: a possible correlation with hypoxia.
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.
Andreadeli
A
A
Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece.
Flemetakis
E
E
Axarli
I
I
Dimou
M
M
Udvardi
M K
MK
Katinakis
P
P
Labrou
N E
NE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000860:Hypoxia / Q000201:enzymology*; D017403:In Situ Hybridization; D000070116:Lotus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005560:Formate Dehydrogenases