The Chl a Carboxylic Biosynthetic Routes: Conversion of Chlide a to Chl a - Chlorophyll Biosynthesis and Technological Applications - page 253

et al. 1980 ). In order to distinguish this enzymatic activity from chlorophyllase it
was named Chl synthetase. Conversion of Chl-GG in vitro to Chl a -phytol by
hydrogenation required the addition of exogenous NADPH. NADH was not a
cofactor (Benz et al. 1980 ). Enzymic hydrogenation of Chl-GG to Chl a-phytol
was inhibited by anaerobiosis (Schoch et al. 1980 ). Substrate specificity
investigations indicated that Chl synthetase requires a chlorin derivative that
contains Mg as a central metal ion. A hydrogenated ring D was mandatory since
Pchlide a with a double bond at position 7-8 of the macrocycle was not a substrate
(Benz and Rudiger 1981 ; Helfrich and Rudiger 1992 ). However, direct esterifica-
tion of endogenous Chlide a with exogenous phytol in the presence of added ATP,
and Mg was also observed in etiolated tissues which led to the proposal that the
conversion of Chlide a to Chl a may follow different biosynthetic routes having
different substrate and cofactor requirements, depending on the stage of plastid
development (Daniell and Rebeiz 1984 ). Subsequently it was determined that in oat
etioplasts, the relative substrate specificities for GG-PP, Phytol-PP and farnesyl-PP
amounted to 6, 3, and 1 respectively (Rudiger 1993 ).
Chlorophyll synthetase is present mainly in the prothylakoid and prolamellar
body of etioplasts (Rudiger 1993 ). Prolamellar body disaggregation and Chlide a
esterification appear to be closely related phenomena. It appears that Chlide
a formed in the prolamellar body can migrate with Pchlide-oxidoreductase to the
prothylakoid membranes during light-dependent dissociation of prolamellar bodies
(Rudiger 1993 ).
10.1.2 Preferential Chlorophyll a Formation
by Esterification of Chlorophyllide
a with Phytol in Green Tissues
Although illumination of etiolated tissues with white light leads to a slow decrease
in Chl synthetase activity (Rudiger 1993 ), the synthetase activity does not disappear
completely, and some activity is still observed in mature chloroplasts (Soll and
Schultz 1981 ). In spinach chloroplasts, the relative substrate specificity for Chlide
a esterification with exogenous GGPP and PhyPP were 1 and 4 respectively a (Soll
and Schultz 1981 ).
In Arabidopsis thaliana a nuclear encoded gene, G 4, was identified which exhibited
homology to the product of the Rhodobacter capsulatus bchG locus which is involved
in the esterification of bacteriochlorophyllide with GG (Gaubier et al. 1995 ). The
relationship between gene G 4and bchG was confirmed by isolation and sequencing
of a corresponding full length cDNA. The gene appears to consist of 14 exons, some of
which were very short. Southern and Northern analyses showed that G 4isasinglecopy
gene and its transcripts were only detected in green or greening tissues.