A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.
Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
Gough
Simon P
SP
Department of Biochemistry, Lund University, Lund, Sweden.
Rzeznicka
Kamila
K
Peterson Wulff
Ragna
R
Francisco
Jose da Cruz
Jda C
Hansson
Andreas
A
Jensen
Poul Erik
PE
Hansson
Mats
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D005612:Freeze Drying; D001467:Hordeum / Q000201:enzymology; D009154:Mutation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011521:Protochlorophyllide / Q000378:metabolism; D011524:Protoporphyrins / Q000302:isolation & purification* / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011524:Protoporphyrins; D002734:Chlorophyll; C033434:protoporphyrin IX monomethyl ester; D011521:Protochlorophyllide; D010105:Oxygenases; C053161:magnesium protoporphyrin monomethyl ester oxidative cyclase