Dw. Bollivar et Si. Beale, FORMATION OF THE ISOCYCLIC RING OF CHLOROPHYLL BY ISOLATED CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS, Photosynthesis research, 43(2), 1995, pp. 113-124
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.