Download Chloroplast is a semiautonomous organelle. Plastid continuity

INTRODUCTION
CHLOROPLAST DEVELOPMENT:
Chloroplast is a semiautonomous organelle.
Plastid
continuity occurs across generations in angiosperms.
Organelles
themselves along with plastid DNA are inherited (Leech, 1984).
In the dividing cells of the meristem, plastid division is
necessary for the maintenance of plastid continuity. In normally
developing plants the youngest plastids are called proplastids
which have few concave and perforated internal membranes.
These
proplastids develop in to mature chloroplasts.
This involves
major internal membrane proliferation and association of these
membranes into grana stacks which is unique to angiosperm
chloroplast.
In contrast to plants grown in diurnal light regime, the
plants grown in the dark for several days contain structurally
complex achlorophyllous plastids, called as etioplasts (Kirk and
Tilney-Bassett, 1967).
During plant growth in dark, the
proplastids undergo abnormal development.
The volume increases
and their internal membranes, proliferate massively and assemble
into a distinctive paracrystalline lattice structure known as the
prolamellar body (Bradbeer 1973).
Illumination of these
etioplasts leads to the development of fully functional
chloroplasts. The light induced synthesis of chlorophyll is the
trigger which ensures the collapse of the regular structure of
the prolamellar body leading to the formation of grana stacks of
the mature chloroplast.
(Boardman and Anderson, 1978).
During
transformation of etioplast to chloroplast, the level of several
enzymes go up including enzymes of chlorophyll biosynthesis goes
up.
Also the number of plastids per cell increases
1973).
(Bradbeer
BIOSYNTHESIS OF 5-AMINOLEVULINIC ACID (ALA):
The first committed precursor of porphyrin biosynthesis
S-aminolevulinic acid, which leads to the synthesis
1
is
of
chlorophylls,
hemes, siroheme and bilins.
ALA synthesis is the
first important regulatory step in porphyrin biosynthesis.
ALA
is synthesised by the condensation of the glycine and succinyl
coenzyme A mediated by the pyridoxalphosphate requiring enzyme,
ALA synthase.
In this reaction carboxyl carbon of glycine is
lost as
co 2
and the reminder is incorporated in to
and, Shemin 1977) .
{Nandi
In higher plants the synthesis of ALA is
carried out by three enzymes, for which
glutamate
ALA.
is the precursor.
the five carbon molecule .
Glutamate is first
ligated to a
glutamate tRNA
by glutamyl tRNA synthetase (Huang et al 1984,
Kannangara et al 1984).
Subsequently it is converted to ALA by
the participation of a dehydrogenase (Weinstein et. al., 1987 )
and an amino transferase (Wang et. al. 1984 ). It is ligated to
tRNA in a reaction identical to the charging reaction in protein
biosynthesis.
Like aminoacyl-tRNA in general, this reaction
requires ATP and Mg2 +.
In the next step tRNA bound glutamate is
converted to a reduced form in a reaction that requires a reduced
pyridine nucleotide.
The product of this reduction has been
characterised as glutamate-1-semialdehyde (Houen, et al 1983) or
its hydrated hemiacetal form {Hoober, et al 1988). Finally, the
positions
carbon
of
the
nitrogen and
intermediate
are
oxo atoms
interchanged
of the
to
reduced
form
ALA.
five
After
demonstration of requirement of RNA for the ALA synthesis (Huang
et al 1984) the tRNA was purified sequenced and characterised as
Glutamate tRNA {Schon et al 1986). Glutamyl tRNA synthetase was
purified from barley chloroplast (Bruyant and Kannangara 1987)
and Chlorella {Weinstein et al 1987). Enzyme Dehydrogenase which
reduces tRNA ligated glutamate has been purified from barley
(Wang et al 1981) .
Amino transferase which converts chemically
synthesized
glutamate-1-semialdehyde to ALA has
been
purified
from barley (Kannangara and Gough, 1978; Wang et al., 1981) and
Chlamydomonas (Wang et al 1984). Not all photosynthetic organisms
use five carbon pathways. For example, Euglena uses both
glutamate and succinate pathway {Weinstein & Beale 1983).
2
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Fig. 1: Protoporphyrin IX biosynthetic pathway.Steps leading to
the the synthesis of proto from ALA is shown in the diagram. a)
ALA dehydratase b)
PBG deaminase
c)
Orogen III
cosynthase d)
Urogen decrboxylase e) Coprogen oxidase f) Protogen oxidase
BIOSYNTHESIS OF PROTOPORPHYRIN IX FROM ALA
The steps in the synthesis of
from
ALA are
very
nonphotosynthetic
protoporphyrin IX (proto IX)
in both photosynthetic as well
Due to the requirement
organisms.
similar
as
of
synthesis of both heroes as well as photosynthetic pigments the
regulation of
complicated
this
which
pathway
is
not
in
the
nonphotosynthetic organisms.
photosynthetic
case
in
organisms
animals
and
is
other
Therefore it is necessary to study
the porphyrin synthesis pathway in plants.
Even though they have
similar sequence of reactions, the regulation and localization of
these enzymes differ
from animals.
In the following text the
individual steps from ALA to proto IX are discussed in detail-f/lg,tl
ALA DEHYDRATASE:
The enzyme catalyses the condensation of two molecules of
ALA to form porphobilinogen (PBG).
This enzyme has been studied.
extensively from a variety of sources.
The animal enzymes
exist as octamers while enzymes from human erythrocytes and beef
liver had molecular weights
(Gibbs et al.,
1985,
&
285
kDa
and
Wilson et al.,
260
kDa
1972) .
respectively
Enzyme from R.
sphaeroides also is an octarner (Gurme et al., 1977) and found to
encode a monomer of 39 kDa (Delaunay et al., 1991).
The enzyme
has been isolated from various plant and algal sources, including
wheat
(Nandi
1969),
radish
and Waygood,
1967),
(Shibata and Ochiai,
tobacco
1977)
al., 1979) and spinach (Liedgens et al.
~rom
1
1
(Shetty and
Chlorella
1980 1 1983).
Miller,
(Tamai et
The enzyme
spinach appears to be a hexamer with a molecular weight of
300 KDa (Liedgen et al., 1980).
A mechanism for the action of ALA dehydratase was proposed
first
by
Shemin 1
197 6
and
demonstrated that ALA formed
that certain inhibitors
of
ALA
with
sodium
et
al.
1977.
They
Schiff base with the enzyme and
(e.g.
for Schiff base formation.
Barnard
levulinic acid)
competed with ALA
Treatment of the enzyme in presence
borohydride
3
led
to
its
irreversible
inactivation.
The site of schiff base formation subsequently has
been identified as a lysine residue in the R. sphoeroides enzyme
(Nandi, 1978) and in animal enzymes (Gibbs and Jordan, 1986).
pH optimum: Enzyme from the plant sources appeared to have an
alkaline pH optimum~ Enzyme from the radish cotyledons showed pH
optimum of 8.0 (Shibata and Ochiai, 1977) while the enzyme from
the spinach leaves showed pH optimum of 8.2 {Schneider, 1970).
Bacterial enzymes also appear to have a pH optimum in the
alkaline range.
Enzyme from R. sphoeroides has a pH optimum of
8.5.
In contrast enzymes from animal showed pH optimum in acidic
range of 6.3-7.1 (Anderson .and Desnick, 1979; Gibbs et al., 1985
; Gurba et al., 1972).
Plant enzymes probably share commonality
more with the bacterial than with the animal system.
Effect of metal ions and metal chelators: Animal and yeast
enzymes require zn 2 +
for their activity (Wilson et al.,
1972;
2
Eight zn + are bound per octamer.
Muthukrishnan et al., 1972) •
Three cy~teines and one histidine are involved in binding the
zinc in a region of the monomer that contains four conserved
cysteine and two conserved histidine residues in the three
species examined (Jordan, 1990) . The zn 2 + do not participate in
binding the substrate molecules (Hasnain et al., 1985); however,
all eight are required for maximum activity (Jordan, 1990).
Enzyme from photosynthetic bacteria required K+ rather than zn 2 +
for
activity
(Van Heyningen and Shemin,
1971).
On the other
hand, the enzyme from R. capsulata did not require any metabolic
cation for its activity (N~mdi and Shemin, 1973).
.Enzyme from
2
2
Spirulina itersonii needed Mg + or Mn + for its activity where as
Zn 2 +, K+, cu 2 + ·had no effect on enzyme activity (Ho and
lascelles, 1971).
In contrast to the animal systems the plant enzymes needed
for their activity.
Enzyme from radish cotyledons was
maximally activated by Mg 2 + as well as .Mn 2 + while K+ was less
Mg 2 +
effective (Shibata and Ochaiai, 1977).
Enzymes from tobacco
leaves as well as radish cotyledons were inhibited by zn 2 + and
4
Fe 2 + (Shetty and MillerJ. 1969; Shibata and Ochiai,
1977).
The
nuclear gene for the ALA dehydratase of pea has been isolated and
sequenced, and found to lack the zn 2 + binding domains
characteristic of the animal enzymes (Li et al., 1991).
But it
was found to contain a distinctive metal-ligand binding domain
based upon aspartate (Boese et al., 1991). This is consistent
with the finding
that plant enzymes require Mg 2 + for their
'
maximum activity instead of zn 2 + (Shetty and Miller 1969; Shibata
and Ochiai 1977).
.
In a recent report on E. coli ALA dehydratase, it was found
that E. coli needs zn 2 + for its catalytic activity where as Mg 2 +
in presence of low amount of zn 2 + can increase the catalytic
activity of the enzyme by decreasing
~
for substrate ALA and
increasing it's Vmax·
It has a binding site distinct from Zinc
2
binding site and Mn + can substitute for Mg 2 + (Jaffe et al.,
1995).
PBG DEAMINASE:
Four molecules of PBG condense to
form uroporphyrinogen.
This reaction is catalysed by PBG deaminase.
Hydroxy methyl
bilane is the initial product of the reaction. In the absence of
uroporphyrinogen cosynthase, the product spontaneously cyclises
to uroporphyrinogen I
(urogen I).
Biologically relevant product
uroporporphyrinogen III (urogen III) is formed in presence of the
enzyme cosynthetase.
The
sources
enzyme PBG deaminase has
(Anedrson and
Desnick,
been purified
1980;
from
animal
Sanovich et al.,
1969),
bacteria (Jordan and Shemin, 1973; Kotler et al., 1987), EUglena
(Williams et al., 1981) , and from plant sources including pea
(Spano and Timko, 1991), wheat germ and spinach leaves (Higuchi
and Bogorad, 1975) Arabidopsis ( Jones & Jordan 1994 ).
It is
also purified from algae (Shioi et al., 1980).
Cloned cDNAs or
genomic sequences encoding PBG deaminase have been isolated from
a variety of sources
like E. coli
5
(Thomas and Jordan,
1986),
Euglena (Sharief et al.,
1989), yeast (Gellerfors et al., 1986)
animal cells {Raich et al., 1986; Chreitien et al., 1988} and pea
(Witty et al., 1993).
Recently, the eDNA for PBG deaminase from
Arabidospsis has been isolated.
of
382
residues,
which
It encodes a precursor protein
can
be
imported
chloroplasts and processed to mature size.
in
to
isolated
It was found to be
encoded by a single gene, which indicated there is only one PBG
deaminase in all plant cells,
which is located in the plastid
(Lim et. al. 1994).
Metal Ions: The PBG deaminase from R. spheroides was inhibited by
However,
metal chelators were found to have no
effect, sulphydryl reagents showed strong inhibition particularly
a strong inhibition was observed with Iodine at 10uM.
soditm
borohydride stimulated the activity
Enzyme from pea chloroplast was
ca 2 + and Mg 2 + were weakly
concentrations
to
inhibit
and
pea
enzyme
inhibitory
Timko,
at
physiological
1991)~
Whereas human
erythrocyte enzyme showed a strong inhibition by Mg 2 +. Mn 2 + was
found
(Spano
(Jordan and Shemin, 1973).
inhibited by Fe 2 +, Mn2 +, zn2 +
at
submillimolar
Significance of this difference is not clear
concentrations.
(Spano and Timko,
1991).
Heat stability: This enzyme from almost all the sources maintain
their activity at temperature ranging from 55-70°C.
The enzyme
from Chlorella regularis is stable even at 75°C for 1 h in the
absence of cofactors or stabilizing ions.
These characters are
comparable to the thermal stability of various enzymes selected
from thermophilic organisms (Shioi et al., 1980}.
Enzyme from R.
spheroides is stable at 60°C in crude, whereas in purified form
it is susceptible to elevated temperatures
(Jordan and Shemin,
1973).
Dipyrromethane Cofactor:
PBG deaminase contain a
methane cofactor (Jordan & Warren,
an
invariant
cysteine was
cysteine
found to
(cys-242)
1987} attached covalently to
in
be present
6
novel dipyrro
E.
coli.
in the
An
equivalent
$~ tU I.Ae'l'\.G€."
primary" Thei heat
stability
of
both
dipyrromethane
the
enzyme
cofactor are
and
the
potentially
explained by
the
large
labile
number
of
protein cofacor interactions revealed in the X-ray structure of
the E. coli PBG deaminse (Louie et.al. 1992 ).
Uroporphyrinogen
III
cosynthetase:
(uroporphyrinogen III synthase)
The
enzyme
cosynthetase
catalyses the formation of uro-
porphyrinogen III from hydroxyl methyl bilane which
product of PBG deaminase activity.
converted
to
biologically
is the
This may be nonenzymatically
inactive
urogen
I.
However,
cosynthetase ensures the formation of only isomer III, which is
biologically active.
Enzyme
has
been
purified
to
homogenity
from
human
gracilis (Hart and
Battersby, 1985) and wheat germ (Higuchi & Bogorad, 1975 ). The
enzyme was found to be thermo labile and activity was enhanced by
Na + and K+. The enzymes PBG deaminase and cosynthetase may be
present as a complex (Tsai et al., 1987).
erythrocytes (Tsai et al.,
1987) Euglena
UROPORPHYRINOGEN DECARBOXYLASE:
Uroporphyrinogen decarboxylase catalyses the decarboxylation
of all four carboxyl residues of uroporphyrinogen to yield
coproporphyrinogen. Enzyme was purified from tobacco leaves (Chen
and Miller 1974).
sources
so
for.
It has not been purified from any other
Animal
sources
from
which
it
is
purified
include human erythrocytes {Deveruneil et al., 1983) and bovine
liver
(Straka and Kushner,
1983).
bacteria Rhodopseudomonas palustris.
It was· also purified froD
The molecular weights
of
enzymes from bacterial and animal sources ranged from 39 to 57
kDa (Koopmann et al., 1986; deVerneuil et al., 1983; Straka and
Kushner,
are
1983) •
accepted
Although all
by
the
enzyme,
four isomers of uroporphyrinogen
aromatic
porphyrins
are
not
decarboxylated (Castelfranco and Beale, 1981).
The discrimation
between
isomers urogen I and urogen III · in conversion into
coproporphyrinogen occurs principally at the first step.
7
Porphyrins especially, oxidation products of the substrates,
inhibited the enzyme (Smith and Francis, 1981).
The activity of Uroporphyrinogen decarboxylase from
oxygen:
tobacco leaves was found to decrease to 57%
in presence of
oxygen. Similar results were obtained with avian erythrocytes
(Tomio et al., 1970)
Enzyme stability: The
tobacco enzyme could maintain 54% of the
activity after being treated with so 0 c for 5 min.
It was found
to be more heat stable than mouse spleen enzyme ( Chen and
Miller, 1974;
Romeo and Lenin, 1971).
It was less stable than
enzyme from R. palustris, which was stable at 60°C for 15 min.
(Koopmann et al., 1986).
pH optimum: Tobacco enzyme was most active at pH 6.5.
From pH
7. 5 to pH 8. o. The enzyme activity decreased sharply to almost
nil~
This pH optimum is quite similar to rabbit erythrocyte
enzyme and R.
palustris
Koopmann et al.,
1986)
enzyme
(Mauzerall
other animal
and Granick,
1958;
sources except for human
erythrocytes which showed a pH optimum of 7.2 (Cornford, 1964).
Co factors: Enzyme prepared from animal or plant sources do not
require any metal ions for their catalytic activity. ca 2 +, Mg 2 +
and zn 2 + were found to have no effect on the enzyme prepared from
rabbit erythrocyte (Mauzerall and Granick, 1958).
Straka and
2
Kushner (1983) found that zn + strongly inhibits the activity
where as Mg2 +, ca 2 + have no effect. But most striking feature of
plant enzyme is that it was not only inhibited by metals like
Fe 2 +, co 2 +, Pb 2 +, Ni 2 + but was also inhibited by Mg 2+ (Chen and
Miller, 1974). The enzyme from avian erythrocyte and tobacco
leaves was stimulated by metal chelators (Tomio et al., 1970).
Bacterial (R. palustris) enzyme was not affected by EOTA (K6opman
et al., 1986).
The enzyme was inhibited by high ionic strength
in both plant and avian erythrocyte Chen and Miller, 1974; Chu
and Chu, 1970).
8
COPROPORPHYRINOGEN OXIDASE:
Oxidative decarboxylation of the propionate side chains on
rings.A and B to give protogen is catalysed by coproporphyrinogen
oxid.-:>e(coprogen oxidase).
In aerobic organisms oxygen is the
sole electron acceptor whereas in anaerobic organisms, a hydride
acceptor such as NADP+ is used (Seehra et al., 1983; Keithly and
Nadler, 1983).
Coprogen oxidase has more substrate specificity
than urogen decarboxylase, and it does not react with coprogens I
or II.
Molecular
properties:
The
enzyme was
tobacco {Hsu and Miller, 1970).
purified
69
fold
fro:m
This is the only plant source
from which the enzyme has been purified. Recently eDNA for
coprogen oxidase was also isolated from soyabean and its primary
structure was determined.
The gene encodes a polypeptide with a
predicted molecular mass of 43 kDa
Coprogen oxidase from bovine liver was
(Madsen et al. 1 1993).
a monomer with molecular
weight of 71.6 kDa (Yoshinaga and Sano, 1980). Yeast enzyme was
found to be a homodimer of molecular weight 70 kDa (Camadro et
al. 1 1986).
Enzyme from mouse liver also was found to be a
homodimer of 70 kDa.
Soybean coprogen oxidase is synthesized
with a putative transit peptide of 67 amino acid residues.
The
full length soybean coprogen oxidase eDNA encodes a protein that
is imported into isolated pea chloroplasts and processed to a
smaller mature form (Madsen et al. 1 1993) .
Expression of the
gene was strongly enhanced in soybean root nodules when compared
to expression in roots and leaves.
It was concluded that the
plant increases the heme production in nodules to meet the demand
for additional heme needed for rhizobia! microsymbionts.
Effect .of neutral deterqents and phospholipids:
extracted from
about 3.6 fold.
bovine
Crude lipids
liver mitochondria activated the enzyme
Activity was also found to increase with
purified phospholipids.
vario~:
Maximum activity was observed with 1-,&
9
lysophosphatidyl choline followed by 1-i(:-i)hosphotodyl choline, 1CI(':~phosphatidyl ethanolamine and ma~y, other phospholipids.
Activity was found to be increased with neutral detergents like
Triton X-100 (0.2%) and Tween 20 (0.2%).
There was no absolute
requirement for
activation
chemical
specificity
(Yoshinaga and Sano,
1980}.
mouse liver enzyme
(Bogorad et al.,
(Camadro et al., 1986).
for
by
lipids
Similar results were obtained for
1989)
and yeast enzyme
No such' studies are carried out in any
of the plant systems so far.
Effect of metal ions and metal chelators:
Purified enzyme from
bovine liver did not show requirement for divalent metal ions.
Addition of ca+ and Mg 2 + (10mM) and Co 2 +, cu 2 +, Fe 2 +, Mn 2 + and
Zn 2 + ( 0.1mM) failed to increase the enzyme activity.
Similarly
the activity was not inhibited by metal chelators (0.1 to. 10mM)
such as
that
no
_;J.-,;J-.
dipyridyl&'phenanthroline.
metals
were
involved
in
the
Thus
it was
active site
of
concluded
coprogen
oxidase (Yoshinaga and Sano, 1980).
On
the contrary
coprogen oxidase from tobacco leaves was
found to be activated by Fe 2 + (0.5 uM), Co 2 + and Mn 2 + (0.1mM).
The enzyme was found to be- inhibited by metal chelators EDTA and
phenanthroline.
Inhibition by EDTA was much higher than that by
0-phenanthroline.
This suggested that some metal ions are in-
volved in coprogen oxidase activity (Hsu and Miller, 1970).
In yeast aerobic coprogen oxidase activity was stimulated in
the presence of divalent ions, whereas anaerobic enzyme activity
had
an
absolute
Polglase, 1974).
is
only one
form
requirement
for
Camadro et al.,
of
a
metal
ion
(Poulson
and
(1986) has reported that there
coprogen oxidase
in yeast
oxygen is absolutely necessary for its activity.
and
molecular
Enzyme from R.
spheroides a photosynthetic bacteria also shows properties
similar to plant enzyme.
The enzyme has aerobic as well as
anaerobic activity.
The anaerobic activity has the requirement
2
for Mg + in addition to nicotinamide nucleotides, ATP and
methionine. It is inhibited by metal chela tors 1, 10 phenanth10
riline and a a' dipyridyl.
But these compounds .have no effect on
aerobic activity (Tait, 1972).
PROTOPORPYRINOGEN OXIDASE: Oxidation of protoporphyrinogen IX
(Protogen) to the fully aromatic porphyrin IX is the only metal
free porphyrin occurring on any of the tetrapyrrolic biosynthetic
pathways.
All the other intermediates are either at the· lower
porphyrinogen
oxidation
level
or
are
metal
complexes.
Protoporphyrinogen is unstable and spontaneously undergoes
oxidation in presence of oxygen and it is enhanced by light •.
The oxidation involves the removal of six hydrogen atoms. This
reaction is carried out by the enzyme protoporphyrinogen oxidase
(protox).
In aerobi_c organisms oxygen is the oxidant, but in
anaerobic organisms the oxidation is achieved by passing
electrons to the electron transport chain (Jacobs and Jacobs,
1979).
Removal of the four hydrogen atoms from the . meso
positions appears to be stereo specific. Protogen oxidase is not
entirely ~pecific for protogen IX, but it is important that there
are no polar groups on ring A or B. Neither urogens nor coprogen
are oxidized by protogen oxidase but protogen XII and
mesoporphyrinogen IX are both substrates for the enzyme.
Proto IX is quite stable towards acids and bases. It is a
rigid planar molecule and can chelate a large variety of metallic
ions at the center of the ring.
It exhibits intense light
absorption in the 400 nm wavelength region and is strongly
fluorescent, emitting light in the region of 630 nm.
These
properties are al related to the attainment of aromaticity, which
is represented by the conjugated system of double bonds in the
prophyrin ring.
It is the aromatic character of the prophyrin
ring that allows. the absorption of light and performance of
photochemistry by chlorophyll.
Protox was purified from barley etioplast and mitochondria.
Enzyme from the two organelles appeared to be identical having
11
molecular weight of 210 kDa.
On an SDS-PAGE single band of 36
kDa was obtained (Jacobs and Jacobs, 1987).
Mg BRANCH OF TETRAPYRROLE BIOSYNTHESIS:
The present investigation does not deal with the Mg
tetrapyrroles, therefore it is reviewed briefly. In higher plants
and some of the photosynthetic bacterial chlorophylls are the
pigments responsible
for
trapping
the
sunlight
for
photosynthesis.
Proto is the branch point for the synthesis of
chlorophylls and hemes.
Insertion of iron (Fe) to the ring by
ferrochelatase gives rise to proto IX heme.
Insertion of
Magnesium leads to the synthesis of chlorophyll.
This Mg
insertion is catalysed by an enzyme magnesium chelatase.
It has
absolute requirement for ATP for its activity.
In cucumber
chloroplasts this enzyme loses its activity on rupturing the
chloroplast
(Richter
and
Rientis,
1982) •
Magnesium
protoporphyrin is converted to methyl magnesium protoporphyrin by
esterification catalysed by the enzyme magnesium protoporphyrin
methyl_transferase. Methyl Mg protoporphyrin undergoes formation
of an isocyclic ring in which the methyl propionates side chain
at position '6' of the macrocycle is joined to the t-mesobridge
of the metalloporphyrin ring forming a five membered ring between
pyrolle ring 'c' and the mesobridge.
This results in the
formation of Mg 2,4 divinyl pheoporphyrin as or divinyl Pchlide.
Vinyl reductase reduces vinyl group at position 4 to ethyl group.
Aronoff et al ( 1971) postulated the existence of parallel
pathways for the formation of chl a, based on the detection of MV
and DV intermediates between MgMPE and Pchlide that accumulated
in mutants of the green alga Chlorella.
Carey and Rebeiz (1985)
classified higher plants as monovinyl (MV) or divinyl(DV) plants.
In barley exogeneously added ov intermediates (proto IX, Mg
proto, and Mg MPE)·were shown to be converted to KV Pchlide at a
point (or points) from proto IX up to (but not including) DV
Pchlide, where as· little or no such conversion occured in
cucumber (Rebeiz et al 1986, Tripathy & Rebeiz, 1986).
Tripathy
12
and Rebeiz (1988) subsequently demonstrated, however, that DV
Pchlide could be reduced to MV Pchlide in barley (but not in
cucumber: at least not on the same time scale) during the
transition from the DV to MV mode of Pchlide synthesis.
Conversion of Pchlide to chlide is the key. step in
chlorophyll biosynthesis. Two hydrogen atoms are added to carbon
7 and 8, trans to each other on ring D. This reduction does not
destroy the aromaticity of the macrocycle.
In higher . plants
light is
necessary for the reduction of Pchlide (Griffiths
1974). Pchlide reductase is the enzyme catalyzing this reaction.
Light induced degradation of Pchlide reductase is one of the
important modes of controlling the chlorophyll biosynthesis. Upon
exposure to light, the enzyme activity, amount of enzyme protein
(Mapelston & Griffiths, 1980 and Santel and Apel, 1981) and the
amount of poly A mRNA from which the protein is translated (Apel,
1981, Batschauer and Apel, 1984} decrease dramatically. The
proteolysis of the reductase occured even when isolated
etioplasts or etioplast membranes from barley were exposed to
continuous light (Kay & Griffiths, 1983, Hauser et al 1984}.
Proteolysis of the enzyme is prevented by the binding of
substrate (Pchlide) to the enzyme (Walker and Griffiths, 1986).
Purified enzyme (Pchlide reductase) from oat seedlings had a
molecular weight of 37kDa (Roper et al 1987).
All the chlorophylls (with the exception of chlorophyll c)
are esterified with a long chain alcohol. It is normally a c-20
alcohol phytol.
This reaction is catalysed by the enzyme
chlorophyll synthetase (Rudiger et al 1980}.
The final product
of the_reaction is chlorophyll a which differs from chlorophyll b
only by the presence of a methyl group in place of the formyl on
ring' ' o f the tetrapyrrole moiety (Beale & Weinstein 1990).
PLASTID ENVELOPE MEMBRANES:
Chloroplast is an organelle which has two limiting membranes
called envelope membranes.
Inside the chloroplast there is a
soluble portion called stroma in which a membranous structure
13
thylakoid is suspended.
Mackender and Leach
(1970)
were the
first to
report
isolation of envelope membranes from intact chloroplasts,
method
employing
centrifugation.
plastid
chloroplast
rupture
and
by a
differential
Some of the most interesting functions of tne
envelope
biog,enesis.
membranes
concern
their
role
in
plastid
The dynamics of the plastid envelope membranes are
important ·for
Inner
osmotic
the
the formation
envelope membrane
is
of
thylakoids during development.
essential
for
the
biosynthesis
plastid components such as glycolipids and prenylquinones.
addition the outer envelope membrane plays a key role
of
In
in the
by nuclear genome
sorting of plastid proteins that are coded
(Douce and Joyard, 1990).
structure
of
the
outer envelope
membrane:
membrane is smooth in outline.
The
outer
envelope
Freeze fracture studies of
chloroplast and etioplast envelopes show that the outer membrane
differs from the inner membrane and from thylakoid in respect to
intra membrane particle distribution (Cline et al., 1985).
membrane has the highest
lipid to
protein ratio
This
(25-30
mg
protein) among plant cell membranes and this is responsible for
its very low density
inner
(1.08 g cm-1}
envelope membrane
possess
frequent
is
folds,
not
always
distinct
invaginate more or less far
(Block et al.,
completely
fr6m
1983).
The
smooth,
but·
thylakioids,
into the stroma.
which
Freeze fracture
studies have shown that the density of intra membrane particles
observed in the inner envelope membrane is higher than in the
outer envelope membrane but lower than in thylakoids
al., 1985).
(Cline et
The lipid to protein ratio of the chloroplast inner
envelope membrane
is high
(about 1-1.2
mg
lipid
fmg
protein)
(Block et al., 1983), corresponding to a density of 1.13 g fcm3
(Cline et al., 1981; Block et al., 1983).
There are contact sites between outer and inner envelope
membranes.
Freeze fracture studies also
lead to
conclusion (Cline et al., 1985; Cremers et al., 1988).
14
the
same
Chua and
Schmidt (1979) proposed that the contact points between the outer
and inner envelope membranes could be sites for protein import
into
chloroplast. At all stages of development the two envelope
membranes are separated by a space of 2-10 nm wide.
There is
little knowledge about the chemical composition and physiological
properties of its content.
from
the cytosol
via
This compartment is freely accessible
the pore protein of
the
outer
envelope
membrane, but not to the plastid stroma' because of limitations
imposed by the specific translocators of the inner envelope
membrane.
Almost
all
the
concentrated in the stroma,
soluble
plastid
proteins
are
only a minute proportion probably
resides in the intermernbrane compartment.
Procedures used for
the separation of outer and inner envelope membrane release the
soluble
proteins
of
the interrnembrane
space
into
the
medium,
together with soluble stromal proteins (Douce and Joyard, 1990).
Some soluble intermembrane proteins may be trapped within the
vesciles
where
osmolarity medium
(1988)
and
Soll
isolated chloroplasts
(Douce and Joyard,
et
al.
1
(1989)
phosphoprotein that could be
are
ruptured
1982).
have
in
low
Soll and Bennett
characterized
located within the
a
64 · kDa
intermembrane
space of the plastid envelope.
Enzymes of chloroplast envelope membranes:
Envelope membranes
isolated from spinach chloroplasts consisted of a
total of at
least 75 polypeptides ranging in molecular weight from 140 kDa to
less than 10 kDa
(Joyard et al.,
1983).
They observed major
bands at 54, 37, 30 1 14 and 12 kDa respectively.
The 14 kDa and
54 kDa protein bands were identified as small and iarge subunits
of
al.
ribulose-1 1 5-bis-phosphate
protein is the phosphate translocator protein
(Flugge
Its
kinetic
(1976)
(Joyard
that the
1981}.
Flugge and Heldt
(RuBPase
found
1
1983).
carboxylase
properties
have
been
30
&
et
kDa
Heldt
investigated
in
reconstituted liposomes (Flugge et al., 1983).
There are several enzymatic activities associated with the
15
chloroplast envelope membrane.
Enzymes of lipid synthesis like
Acyl ACP; snglycerol-3-phosphate acyltransferase, Acyl-ACP:monoacylglycerol-3-phosphate acyltransferase; are identified in the
envelope membranes (Joyard and Douce, 1977).
Also a lipid
biosynthesis enzyme UDP-galactose:diacylglycerol galactosyl
transferase is present in the inner envelope membrane (Block et
al, 1983). It has been partially purified from Spinach chloplasts
(Coves et al 1986). This enzyme is used as a marker enzyme for
inner envelope membrane of the chloroplast.
Enzymes of
flavahoid
biosynthesis
are
also
~ssociated
with
envelope
membranes
(Costes et al., 1986; Soll et al.,. 1980).
A DCCD
insensitive Mg 2 + depende~t ATPase was also associated with
plastid envelope membranes (Douce et al., 1973). It has been
purified f~om spinach chloroplasts (Nguyen and Siegenthaler,
198S) and Pea (McCarty and Selman, 1986).
Recently Pchlide reductase was shown to be present in outer
envelope membranes, protox was
detected in the envelope membrane
(Joyard et al., 1990; Martinge et al., 1992) and Mg chelatase was
also found to be present in inner envelope membrane of the
chloroplast (Fuesler et al., 1984).
These findings connect the
envelope membranes with porphyrin biosynthesis.
LOCALIZATION OF TETRAPYRROLE BIOSYNTHESIS ENZYMES:
One of the ways in which organisms regulate their metabolic
activities is through compartmentalization of various metabolic
activities. Compartmentalisation ensures functional diversity and
versatility to the cell. Compartmentalisation of enzymes is one
of the most important mode of regulation.
Therefore it is
important to know the localization of the enzymes along with
other aspects of regulation.
The chloroplast can be .divided into several compartments.
· Each compartment gives an enzyme a particular kind of environment
which is different from rest of the compartments. Roughly the
16
chloroplast can be divided into a)
inner envelope membrane c)
outer envelope membrane b)
stroma d)
thylakoid membranes e)
envelope inter membrane space and f) thylakoid lumen.
Enzymes of heme synthesis in animals were
found to be
localised in two cellular compartments; the mitochondria and the
cytoplasm.
The synthesis
(Jordan, 199-0}.
of ALA takes
place
in mitochondria
The product ALA, passes out of the mitochondria
and into the cytoplasm where the next four enzymes are found as
soluble proteins.
The product of these enzymatic steps, coprogen
III, returns to the mitochondria where it is converted· by three
enzymes coprogen oxidase,
convert it
to proto
protogen oxidase and ferrochelatase,
heme (Moore, 1990).
In animals, the enzyme
coprogen oxidase is an easily dissociable extrinsic protein
(Grandchamp et al.,
1978 & Elder and Evans,
1978),
whereas
protogen oxidase (Deybach et al., 1985} and ferrochelatase (Jones
and Jones, 1969} are firmly_bound intrinsic proteins.
In higher plants,
the
localisation
of
tetrapyrrole synthesis is not as conclusive as
animals.
to
the
enzymes
of
in the case of
It was demonstrated that chloroplasts alone were able
convert
glutamate
to
chlorophyll
a.
Therefore,
the
chloroplast must contain all the necessa-ry enzymes for this
process
(Fuesler et a1.,
1984}.
It was also demonstrated that
glutamate and other 5-carbon compounds, rather than glycine, were
exclusively used to form the heme moieties of mitochondrial
cytochrome oxidase in the red alga Cyanidium caldarium (Weinstein
and Beale,
1984}
and etiolated maize
(Schneegurt and Beale,
1986).
The last two enzymes of heme synthesis, protox (Jacobs and
Jacobs,
1984;
1987)
and
ferrochelatase
(Perra
and
Lascelles,
1968, Little and Jones, 197'6) have been detected in mitochondria
and chloroplasts.
Each enzyme was found to be associated with
membranes in the organelles.
However coprogen oxidase was shown
to be present in chloroplast and
17
not in mitochondria (Smith et
Therefore protogen IX or proto IX is likely to be
al., 1993).
transported into mitochondria from the chloplast for the
mitochondrial heme syntheis. Recently,· it has been shown by
herbicide binding studies that the enzyme protox is localised in
the envelope membrane
(Martinge et al.,
1992).
Smith
(1988)
studied the distribution of two of these enzymes, ALA dehydratase
and PBG deaminase, in pea and in the spadices of Arum, where the
synthesis of mitochondrial heme is predominant. In both of these
plants,
the
distribution
of
these
enzymes
into
various
subcellular fractions parallelled the distribution of a soluble
chloroplast stromal marker enzyme, but not marker enzyme by the
cytoplasm or mitochondria.
These results were consistent with an
exclusive plastid location for these two enzymes.
These results
were further confirmed by isolation of gene for PBG · deaminase
from the nuclear genome, and import of the in vitro translated
precursor protein into the chloroplast of Arabidopsis (Lim et
al., 1994).
There is only one gene which encodes for the enzyme
PBG deaminase (Lim et al., 1994) and it is localised in
the
chloroplast.
Intraplastidic Localization of Enzymes of Protporphyrin IX
Biosynthesis: smith and Rebeiz (1979) had concluded earlier that
all the enzymes for the conversion of ALA to prot IX were soluble
stromal enzymes in cucumber whereas enzymes of the magnesium
branch were membrane bound. Castelfranco et al. (1988} supported
a stromal location for PBG deaminase in cucumber, however Nasri
et al.
(1988) found that about two-thirds of the activity of ALA
dehydratase was soluble and the remainder was membrane bound in
the etiochloroplasts of radish.
Lee et al (1991) carried out the
osmotic lysis of carefully purified etiochloroplasts of cucumber
and reported that nearly 90% of the activity of enzymes
converting ALA to proto IX remained with the membrane faction.
Virtually all the activity was released into the supernatant
faction by a high speed homogenisation, indicating that these
enzymes were associated only loosely with the membrane, perhaps
18
as an extrinsic enzyme complex (Lee et al.,
1991) •
Thus,
the
question of localisation of enzymes of ALA to proto IX conversion
is far from clear.
Therefore, in the present investigation,
attempts were made to enhance our knowedge on localization of
enzymes leading to proto IX synthesis.
REGULATION OF TETRAPYRROLE BIOSYNTHESIS:
Hemes are present in etiolated leaves and chlorophyll is
absent,
although
present.
After
small
exposure
amounts
to
of
light,
protochlorophyllide
the
pr'otochlorophyllide
immediately photo converted to chlorophyllide.
period,
a
phase
of rapid
After a
chlorophyll accumulation
Chlorophyll accumulation is complete after 48 h
Weistein, 1990).
are
is
lag
begins.
(Beale and
There are three key steps at which the chlorophyll and heme
biosynthetic pathway are regulated. First, at the level of syn~
thesis of the first precursor of the pathway ALA; second, at the
level of . metal chelation and third,
at conversion of protoch-
lorphyllide to chlorophyll ide in the chlorophyll branch.
ALA
formation from glutamate (i. e., 5 carbon pathway) is exerted at
the dehydrogenase step through
feed back inhibition and
induction/repression. In some species, end product inhibition of
the glutamyl-tRNAglu level may also occur.
Heme is the potent
inhibitor of ALA formation in intact plastids (Beale, 1990).
Mg
protoporphyrin also inhibits ALA synthesis in intact plastids.
Ability to synthesize ALA increased by light pretreatment (Huang
and Castelfranco, 1989) indicating the phytochrome mediated
synthesis of enzymes.
Insertion of the. central metal ion into the protoporphyrin
is the step that controls the flux of porphyrins to either hemes
or chlorophylls.
Enzyme ferrochealatase is. inhibited by heme as
well as by Mg protoporphyrin (Little and Jones, 1976) .
Mg
chelatase requires ATP.
In intact plastids the activity of Mg
chelatase was effectively inhibited by exogenous Pchlide and
Chlide (Pardo et al., 1980).
19
A model was proposed by Beale and Weinstein
(1990)
to
account for the rapid response of chlorophyll and ALA synthesis
to
light,
while
accumulating
the
need
for
photosynthetic and nonphotosynthetic precursors.
is
heme
in
both
ALA synthesis
controlled by substrate supply and -feed back inhibition by
heme
at
possibly
two
enzymatic
steps,
subsequent reduction of glutamyl-tRNA.
the
formation
and
Ferohelatase is probably
subject to product inhibition by heme and possibly by Mg proto. as
well.
In dark, modulation of ALA synthesis is controlled by the
level of a heme pool which constantly turns over.
Pchlide also
accumulates in the dark which serves as a feed back inhibitor of
Mg chelatase.
Th_is forces the proto IX
through the Fe branch,
thus contributing to a constant supply of heme.
heme, Pchlide does not turn over in the dark.
In contrast to
In the light, the
bound Pchlide is immediately photoreduced, thus making the
on the reductase
sites
free for new Pchlide molecules. The lowering of
free Pchlide concentration, in turn relieves the inhibition of Mg
chelatase.
Activation of Mg chelatase diminishes the
flux of
proto IX through the Fe branch, causing a depletion of the heme
pool, thereby releasing the inhibition of ALA synthesis.
Regulation of Intermediate Steps of
Cloned
dehydratase
cDNAs
and
genomic
(Boese et al.,
Tetrapyrrole Biosyntheis:
DNA
1991;
Li
fragments
et al.,
encoding
1991)
ALA
and PBG
deaminase (Shashidhara and Smith, 1991; Witty et al., 1993) have
now been isolated from several plant and algal species and a eDNA
for coprogen oxidase has also been isolated from soybean (Madsen
et al. , - 1993) .
All the genes isolated possessed an N-terminal
transit peptide coding regions.
synthesis abundance and
activity of ALA dehydratase and PBG deaminase are regulated by
light and cell type (Smith, 1988; Spano and Timko, 1991; Boese et
al.,
two
1991; Shashidhara and Smith 1991).
enzyme
activities
appear
to
The abundance of these
be
subject
more
to
transcriptional control in early development, where as at later
20
developmental stages,
the regulatory influence of one or more
posttranslational process ( eg. enzyme activation) seems to
predominate (Spano and Timko, 1991; Boese et al., 1991, Witty et
al., 1993) .
In Euglena like in higher plants, photo regulation
of the enzyme activities catalysing the intermediate steps of
chlorophyll
and
heme
formation
predominantly occurs
post-
transcriptionally (Shashidhara and Smith, 1991).
Thus,
addressed
it is clear that, there are several questions to be
in porphyrin synthesis.
One such area is the
regulation of enzymes of intermediates of ALA to protoporphyrin
IX and also the intrachloroplastidic location of these enzymes.
The
steps . leading
from
5-amino
levulinic
acid
to
protoporphyrin IX synthesis is the topic of the present study.
The broad aims of the study were:
1. to determine the intrachloroplastic localisation of the
enzymes of ALA to proto IX synthesis.
2. to determine the regulation of enzymes of protoporphyrin
synthesis from ALA.
21