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6, 1988
Polyhydroxybutyrate: an Intriguing
Biopolymer
Edwin A. Dawes
INTRODUCTION
O--CH--CH2--C-~
537
0144-8463/88/1200-0537506.00/0O 1988 Plenum Publishing Corporation
538 Dawes
the same rate and thus are diverted to the reductive step of polymer synthesis
(Senior and Dawes, 1973). This hypothesis received support from measurements
of the intracellular N A D H / N A D § ratio during transition from a nitrogen to an
oxygen limitation. The ratio increased dramatically on imposition of the oxygen
limitation but then, as PHB synthesis commenced, decreased to a lower level but
one which was rather higher than that recorded for the original nitrogen-limited
steady state (Jackson and Dawes, 1976).
Investigations of carbohydrate metabolism in A . beijerinckii showed the
operation of the Entner-Doudoroff pathway with terminal oxidation via the
tricarboxylic acid cycle. We found that metabolism of glucose was subject to
regulation at three points as a consequence of an oxygen limitation. First, at the
g~ucose 6-phosphate dehydrogenase step: this is an allosteric enzyme inhibited by
ATP and, even more powerfully, by N A D P H and NADH. Second, citrate
synthase is subject to inhibition by N A D H , an effect which can be overcome by
1 mM AMP, in keeping with David Weitzman's general observation relating to
Gram-negative bacteria (Weitzman and Jones, 1968). The third enzyme under
control is isocitrate dehydrogenase, inhibited by N A D P H , N A D H and ATP.
Thus in the tricarboxylic acid cycle there are two points of control: citrate
synthase and isocitrate dehydrogenase. The cycle is, of course, generating
intermediates for biosynthesis as well as effecting oxidation and energy gener-
ation. Furthermore, the organism is fixing nitrogen, a process which requires both
reducing power and a significant input of energy, estimated at some 15 ATP per
mole N2 reduced.
Thus, when an oxygen limitation is imposed and the N A D H / N A D § ratio
rises, inhibition of citrate synthase and isocitrate dehydrogenase will occur, the
tricarboxylic acid cycle will slow down and this could have serious effects on the
growth of the organism. However, PHB synthesis, by effectively "mopping up"
some of the excess reducing power, redresses the N A D H / N A D + balance and
relieves the inhibition, enabling the cycle to operate at a rate faster than would
otherwise have been possible (Senior and Dawes, 1971).
Inhibition of citrate synthase, the initial enzyme of the tricarboxylic acid
cycle, leads to an accumulation of acetyl-CoA, the precursor of PHB, and to link
up these two segments of metabolism it is now necessary to consider the pathway
of polymer synthesis.
Early experiments had ruled out the involvement of an acyl carrier protein in
PHB synthesis and had shown that CoA esters were intermediates (Ritchie and
Dawes, 1969). The route of synthesis from acetyl-CoA established involves three
enzymes: 3-ketothiolase, acetoacetyl-CoA reductase and PHB polymerase. Two
molecules of acetyl-CoA are condensed by 3-ketothiolase, with the release of
CoA, to yield acetoacetyl-CoA, which is then reduced by the reductase to
D(-)-3-hydroxybutyryl-CoA (Ritchie et al., 1971) which, in turn, is polymerized
with the release of CoA. The biosynthetic pathway is controlled by 3-ketothiolase
activity. We discovered that the reversible reaction catalysed by this enzyme is
inhibited in the condensation direction by free CoA, while in the direction of
thiolysis high concentrations of acetoacetyl-CoA inhibit but this effect is reversed
by increasing the concentration of the other substrate, CoA (Senior and Dawes,
540 Dawes
1973). The Km for acetyl-CoA is quite high (0.9 raM). Similar behaviour had been
shown for the 3-ketothiolase of Hydrogenomonas eutrophus H16 (Alcaligenes
eutrophus) by Oeding and Schlegel (1973).
We found that acetoacetyl-CoA reductase was five-fold more active with
N A D P H than with N A D H throughout purification. It is a typical thiol enzyme
sensitive to the usual inhibitors. The final enzyme of synthesis is the polymerase
or synthase which is associated with the membrane of the polymer granule. This
enzyme is of crucial importance and the subject of intensive interest although, at
the present time, relatively little has been published. It is a fascinating system
involving a reaction in which soluble components of the cytoplasm react at a
surface to give a water-insoluble product which accumulates in presumably an
extremely hydrophobic environment within the granule. Griebel and Merrick
(1971) proposed that polymerization is a two-stage reaction involving the
formation of an acyl-enzyme intermediate via a functional thiol group on the
enzyme:
D( - )-3-hydroxybutyryl-CoA + Synthase-SH--*
3-hydroxybutyryl-S-Synthase + CoA
3-Hydroxybutyryl-S-Synthase + PHB primer (n units)
PHB primer (n + 1 units) + Synthase-SH.
Degradation of PHB occurs via a pathway which does not involve CoA
esters. In A. beijerinckii a depolymerase associated with the polymer granule
membrane initiates hydrolysis to yield free D ( - )-3-hydroxybutyrate, which is
then oxidized to acetoacetate by an NAD-specific dehydrogenase. This enzyme is
competitively inhibited by NADH, pyruvate and 2-oxoglutarate; N A D P H has no
effect. We discovered that the acetoacetate is next converted to acetoacetyl-CoA
via the action of an acetoacetate : succinate CoA transferase, an important finding
because it meant that PHB metabolism is a cyclic process, acetoacetyl-CoA
serving as a precursor of PHB synthesis and also as a product of its degradation
(Fig. 1).
It is of interest that while A. beijerinckii and Ale. eutrophus both possess an
acetoacetate:succinate CoA transferase, in Zoogloea ramigera acetoacetate is
esterified with CoA by a CoA-synthetase reaction employing ATP (Tomita et al.,
1983).
Acetoacetate + CoA + ATP---~ acetoacetyl-CoA + AMP + PPi
PYRUVATE
k _jt
Acetoocetyl- SCoA
I
I
// :..o,~," \N~ J
NADH
2- Oxoglutarate
ACETOACETATE
~NAOH /'
D(-)-3- H Y O R O X Y B U T Y R Y L - S C O A
O[-)-3-HYDROXYBUTYRATE [POLY-@-HYOROXYBUTYRATE I
Recently Doi et al. (1986) in Japan have examined the route by which the
hydroxyvalerate unit is derived when Alc. eutrophus is incubated with [2-
13C]acetate or [1-13C]propionate in the absence of a nitrogen source. Polymer
isolated from the organism after 48h incubation was analysed by ~3C N.M.R. in
each case and the results accorded with PHB synthesized from [2-~3C]acetate
being labelled in the 2 and 4 positions of the HB units, while in the case of the
[1-~3C]propionate substrate the following scheme was proposed to account for the
labelling pattern:
COPOLYMER
CH3COO- ~ CH3CH.CH2COO-
I
OH
The random copolymer was labelled only in the C-3 of the HV units. The HB
units are unlabelled because the label is lost as CO2 in the conversion of
propionate to acetate.
We were interested to know whether any turnover of polymer occurs in the
bacterial cells. Thus we grew Alc. eutrophus on [U-14C]glucose in a chemostat
and, after a steady state was attained, the labelled substrate was replaced by
unlabelled glucose and the loss of radioactivity from the whole cells and from
isolated PHB was compared. Both mirrored the washout rate and we concluded
that no significant turnover was occurring.
I % ,o o I -
/ Methanol . " ~ ' l - - i ' n - r-I- 1 - 1 ~
o -l-I /
6
o
"5
..--o //\ f l
Fig. 2. Accumulation of PHB and PHV by Pseudomonas extorquens growing in batch culture with
methanol as carbon source and limiting ammonium as nitrogen source. At the time of exhaustion of
the ammonium (indicated by solid arrow), valeric acid was added to the culture.
REFERENCES