Maize uroporphyrinogen III methyltransferase: overexpression of the functional gene fragments in Escherichia coli and one-step purification.
S-Adenosyl-L-methionine: uroporphyrinogen III methyltransferase (SUMT), a key regulatory enzyme, converts uroporphyrinogen III to precorrin-2 in the porphinoids biosynthesis. In this study, the mature SUMT was signified that the maize SUMT precursor encoded by the open reading frame of maize SUMT cDNA was deleted the first 91 amino acids constituting the postulated signal peptide. Several mature SUMT fusion and deletion mutants were conducted. It actively expressed in Escherichia coli that the mature SUMT, or the truncated one deleting the C-terminal extra 52 amino acids based on SUMT sequence comparisons. On the contrary, it expressed as an inclusion body in E. coli that the mature SUMT fusion mutant, the SUMT precursor, or the mature SUMT deleting the N-terminal 36 amino acids including glycine-rich region involved directly in SAM binding. The purified His6-tagged mature SUMT was homodimer with a molecular weight of 34 kDa, as shown by SDS-PAGE, 52 kDa using gel-filtration chromatography, and 79 kDa by dynamic light scattering assay. Red fluorescent compounds were associated with the recombinant mature SUMT which were identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis. This association slightly altered the protein secondary structure confirmed by circular dichroism assay.
Fan
Jun
J
Hefei National Laboratory of Physical Sciences at Microscale, Key Laboratory of Structural Biology of Chinese Academy of Science, 96 Jinzhai Road, Hefei, Anhui 230026, PR China.
Wang
Deqiang
D
Liang
Zhi
Z
Guo
Min
M
Teng
Maikun
M
Niu
Liwen
L
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D016296:Mutagenesis; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D003313:Zea mays / Q000201:enzymology*
D029968:Escherichia coli Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase