Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803

被引:26
|
作者
Shepherd, M [1 ]
Reid, JD [1 ]
Hunter, CN [1 ]
机构
[1] Univ Sheffield, Dept Mol Biol & Biotechnol, Robert Hill Inst Photosynth, Sheffield S10 2TN, S Yorkshire, England
关键词
chlorophyll biosynthesis; methylation; porphyrin;
D O I
10.1042/BJ20021394
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX, propionate side chain to form magnesium (MgP) at the C-6 protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases. Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP. Initial rate studies showed that the reaction proceeds via a ternary complex. Product (S-adenosyl-L-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further. SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD. This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme - or a ChlM-MgD complex. Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.
引用
收藏
页码:351 / 360
页数:10
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