title: Cloning, expression, characterization and mutational analysis of the tfdA gene from Cupriavidus campinensis BJ71.
abstract: 2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. Previous studies focused on the tfdA gene in Ralstonia eutropha JMP134 isolated in Australia. In this study, a new tfdA gene was cloned from Cupriavidus campinensis BJ71, an effective degrading bacteria from China, based on the iCOnsensus-DEgenerate Hybrid Oligonucleotide Primers (iCODEHOPs) protocol, combined with high-efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR). The open reading frame of 861 bp encoded a putative 287 amino acid protein with a theoretical molecular mass of 32.32 kDa. The gene was overexpressed in Escherichia coli BL21 (DE3) and the purified TfdA showed optimal activity at pH 6.75 and 30 °C. This enzyme was more thermostable and it could use 3-hydrocinnamic acid as substrate, with a similar enzyme activity compared with 2,4-D. TfdA and its variants were created as maltose-binding protein (MBP) tagged fusion proteins to examine the roles of putative substrate-binding residues. The MBP-N110A, MBP-V198A and MBP-R207K proteins showed decreased k cat and increased Km, and MBP-R278A was inactive, suggesting these residues may affect 2,4-D binding or catalysis.
authors: Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guiyang, 550025, China, hanlizhen11@163.com.
authors: Lizhen
authors: L
authors: Han
authors: Yanbo
authors: Y
authors: Liu
authors: Cuicui
authors: C
authors: Li
authors: Degang
authors: D
authors: Zhao
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D049921:Cupriavidus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010666:Phenylpropionates / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010666:Phenylpropionates; C035253:3-phenylpropionic acid; D006899:Mixed Function Oxygenases; C084087:2,4-dichlorophenoxyacetate-alpha-ketoglutarate dioxygenase