An enzyme-coupled continuous spectrophotometric assay for magnesium protoporphyrin IX methyltransferases.
The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are unsuitable for analysis of the catalytic properties of ChlM. Here we report the successful development of a coupled, continuous spectrophotometric assay to measure the activity of ChlM. The product of the methyltransferase reaction, S-adenosyl-l-homocysteine (SAH), is converted into adenine and then hypoxanthine by the recombinant coupling enzymes SAH nucleosidase and adenine deaminase, respectively. The appearance of hypoxanthine results in a decrease in absorbance at 265nm. The utility of this assay was shown by the characterization of ChlM from the cyanobacterium Synechocystis sp. PCC 6803. Kinetic parameters obtained support data acquired using the discontinuous HPLC-based assay and provide further evidence for the stimulation of ChlM by the H subunit of magnesium chelatase (ChlH).
McLean
Samantha
S
Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. s.mclean@sheffield.ac.uk
Hunter
C Neil
CN
D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D000619:Aminohydrolases / Q000378:metabolism; D001681:Biological Assay; D000458:Cyanobacteria / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015394:Molecular Structure; D011524:Protoporphyrins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology; D014169:Transformation, Bacterial
D011524:Protoporphyrins; D011994:Recombinant Proteins; D019271:Hypoxanthine; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C028025:protoporphyrin IX; D008780:Methyltransferases; C060234:magnesium-protoporphyrin methyltransferase; D000619:Aminohydrolases; C018774:adenine deaminase; D000225:Adenine