title: A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.
abstract: 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.
authors.affiliation: Department of Biochemistry, Lund University, Lund, Sweden.
authors.forename: Simon P
authors.initials: SP
authors.lastname: Gough
authors.forename: Kamila
authors.initials: K
authors.lastname: Rzeznicka
authors.forename: Ragna
authors.initials: R
authors.lastname: Peterson Wulff
authors.forename: Jose da Cruz
authors.initials: Jda C
authors.lastname: Francisco
authors.forename: Andreas
authors.initials: A
authors.lastname: Hansson
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.forename: Mats
authors.initials: M
authors.lastname: Hansson
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D011524:Protoporphyrins; D002734:Chlorophyll; C033434:protoporphyrin IX monomethyl ester; D011521:Protochlorophyllide; D010105:Oxygenases; C053161:magnesium protoporphyrin monomethyl ester oxidative cyclase