FORMATION OF THE ISOCYCLIC RING OF CHLOROPHYLL BY ISOLATED CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chi biosynthesis, S-adenosyl-L-methionine:Mg-protoporphyrin IX meth yltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cycl ase. A double mutant strain of C. reinhardtii was constructed which ha s a cell wall deficiency and is unable to form chlorophyll in the dark . Dark-grown cells were disrupted with a BioNeb nebulizer under condit ions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation . The purified chloroplasts were used to define components required fo r the biosynthesis of Mg-2,4-divinylpheoporphyrin a(5) (divinyl protoc hlorophyllide) from Mg-protoporphyrin TX. Product formation requires t he addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-L-me thionine:Mg-protoporphyrin IX methyltransferase which produces Mg-prot oporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl es ter that is generated in situ is the substrate for Mg-protoporphyrin I X monomethyl ester oxidative cyclase. The reaction product was identif ied as Mg-2,4-divinylpheoporphyrin a(5) (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired r everse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a(5 ) formation by the coupled enzyme system required O-2 and was stimulat ed by the addition of NADP(+), an NADPH regenerating system, and S-ade nosyl-L-methionine. Product was formed at a relatively steady rate for at least 60 min.