Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078.
The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His(6)-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414 mU mg(-)(1) protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3A resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70-73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.
Kan
Shu-Chen
SC
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Liu
Jai-Shin
JS
Hu
Hui-Yu
HY
Chang
Chia-Ming
CM
Lin
Wei-De
WD
Wang
Wen-Ching
WC
Hsu
Wen-Hwei
WH
D000595:Amino Acid Sequence; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D013940:Thymidylate Synthase