THE CHLOROPHYLL BIOSYNTHETIC ENZYME MG-PROTOPORPHYRIN IX MONOMETHYL ESTER (OXIDATIVE) CYCLASE - CHARACTERIZATION AND PARTIAL-PURIFICATION FROM CHLAMYDOMONAS-REINHARDTII AND SYNECHOCYSTIS SP PCC-6803
Dw. Bollivar et Si. Beale, THE CHLOROPHYLL BIOSYNTHETIC ENZYME MG-PROTOPORPHYRIN IX MONOMETHYL ESTER (OXIDATIVE) CYCLASE - CHARACTERIZATION AND PARTIAL-PURIFICATION FROM CHLAMYDOMONAS-REINHARDTII AND SYNECHOCYSTIS SP PCC-6803, Plant physiology, 112(1), 1996, pp. 105-114
A universal structural feature of chlorophyll molecules is the isocycl
ic ring. This ring is formed by the action of the enzyme Mg-protoporph
yrin IX monomethyl ester (oxidative) cyclase, which catalyzes a comple
x reaction in which Mg-protoporphyrin IX monomethyl ester is converted
to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyr
in a(5)), with the participation of NADPH and O-2. Cyclase activity wa
s demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and ext
racts of Synechocystis sp. PCC 6803. The yield of the reaction product
was increased by the addition of catalase and ascorbate or isoascorba
te to the incubation mixture. These compounds may act by preventing de
gradation of the tetrapyrroles by reactive oxygen species. Cyclase act
ivity from C. reinhardtii was not inhibited by the flavoprotein inhibi
tor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In c
ontrast, cyclase activity in extracts of C. reinhardtii and Synechocys
tis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that non
heme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii c
hloroplasts was associated with the membranes, and attempts to further
fractionate or solubilize the activity were unsuccessful. In contrast
, cyclase in Synechocystis sp. PCC 6803 extracts could be separated in
to soluble and membrane components, both of which were required for re
constitution of activity. The membrane component retained activity aft
er it was solubilized by the detergent n-oetyl-beta-D-glucopyranoside
in the presence of glycerol and Mg2+. The solubilized membrane compone
nt was purified further by dye-affinity and ion-exchange chromatograph
y.