FEMS Microbiology Letters 34 (1986) 123-127
Published by Elsevier
123
FEM 02363
Hypothesis
Evolution of the glyoxylate bypass in E s c h e r i c h i a c o l i - - an
hypothesis which suggests an alternative to the Krebs cycle
(Glyoxylate oxidation cycle; growth on acetate; flux through central metabolic pathways)
W.H. Holms
Department of Biochemistry, Universityof Glasgow, GlasgowG12 8QQ, U.K.
Received 9 November 1985
Accepted 11 November 1985
.,~1.06
1,03
INTRODUCTION
When aerobic heterotr0phs such as Escherichia
coli grow on single carbon sources they convert
these into components of the central metabolic
pathways which perform two functions: (1) the
central pathways generate the precursors from
which all the monomers of new cell material are
made; (2) the central pathways provide the energy
sources (PEP, the proton motive force, ATP and
NADPH, H ÷) which are used for substrate uptake, biosynthesis and growth.
An overall view of the central pathways can be
constructed (Fig. 1) from the known reactions [1]
of the metabolic pathways involved - - glycolysis,
the l~ntner-Doudoroff pathway, the Krebs cycle
and the various reactions interconnecting these
pathways. The selection of reactions actually used
on any given carbon source depends largely on its
point of entry into the central pathways. From the
monomeric composition of E. coli [2] and the
biosynthetic routes leading to the monomers [1] it
is possible to compute the drain of compounds
from the central pathways (Fig. 1) for biosynthesis
of new cells [3]. It is most convenient to express
these as retool. (g dry wt biomass formed)-1. If
the input of carbon source is known, it is then a
question of simple arithmetic to calculate the
FDPJ t
y
O4"!
T,P6 ~
-
J
CIT
cycle
)/L_&
/
]
~ 2PG
Fig. 1. Outputs to biosynthesis from the central pathways.
Double-shafted arrow, outputs to biosynthesis: retool. (g dry wt
biomass formed)-~. Ac-CoA, acetyicoenzyme-A; CIT, citrate;
DHAP, dihydroxyacetone phosphate; DPG, diphosphoglycerate; F6P, fructose 6-p~osphate; FDP, fructose 1.6 bisphosphate; G6P. glucose 6-phosphate; IsoCIT. isocitrate;
KDPG, 2-keto 3-dooxy 6-phosphogluconate; MAL, malate;
OAA, oxaloacetate; OGA, 2-oxoglutarate; PEP. phopshoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate;
6PG, 6-phosphoglnconate; PP. pentose phosphate; PYR,
pyruvate; TP. triose phosphate. Circled numbers: 1, fructose
1,6- bisphosphatase; 2, phosphofructokinase; 3, phosphotransferase for substrate uptake; 4, pyruvate kinase; 5, PEP
synthase; 6, PEP carboxylase; 7, PEP carboxylase; 7, PEP
carboxykinase.
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
124
0.72
JP~P~
0.2,, ,A/
~
/
f
-C,T
'.o'= . ; 2
Acetate
Input
T
1
o
NAI~I
0,45~'
CO; i ~IADPH
Fig. 2. Flux through the central pathways during growth on acetate. Abbreviations as in Fig. 1 and: F U M , fumarate; ICDH, isocitrate
dehydrogenase; 1CL, isocitrate lyase; SUC, succinate. N u m b e r s in boxes are net fluxes as mmol substrate processed . (g dry
w t ) - i. h - 1. E. coil ML308 was grown aerobically in a defined medium [7] with acetate (30 raM) as sole source of carbon [8]. Growth
was measured as apparent absorbance (420 nm) in a Unicam SP-30 spectrophotometer calibrated for bacterial dry weight. Acetate in
the cultures was measured [7] and its concentration plotted against growth which gave a strainght line the slope of which is acetate
utilisation [mmol acetate.(g dry w t ) - l ] . This method of using many determinations from several cultures to measure yield gave very
reproducible results (linear correlation coefficients of the plots fell within the range 0.995 to 0,999). The acetate input was 46.89
m m o l , g -1 and the growth rate (/x) was 0.43 h -1 and these data were used to compute the flux of acetate through the central
pathways to biosynthesis and CO:.
throughput (mmol. (g dry wt) - i ) of each enzyme
in the 'central pathways required to generate the
precursors for biosynthesis and to oxidise the balance of input carbon to carbon dioxide. Further, if
the growth rate (/~; h -~) for the particular carbon
source is measured, throughputs for each enzyme
can be expressed as net fluxes in terms of mmol
substrate processed. (g dry wt)- ] • h - ]. In this way
the net fluxes through the enzymes of the central
pathways have been calculated for E. coli ML308
growing on acetate (Fig. 2). The effects of futile
cycles [4] and fluxes to maintain pools of intermediates [5] are small and are discussed elsewhere
[31.
2. THE GLYOXYLATE BYPASS
Growth on acetate depends on the glyoxylate
bypass and the Krebs cycle [6]. In this system the
enzymes of the anaplerotic sequence (isocitrate
lyase (ICL) and malate synthase) convert acetylCoA to malate. The surplus malate gives
oxaloacetate directly which, in turn, leads to
oxoglutarate and also, via PEP-carboxykinase, to
PEP and thence the other precursors required for
biosynthesis [6] (Fig. 2). Note that there is another
route from malate to pyruvate by malic enzyme
(not shown). While the glyoxylate bypass tarps
some reducing power, the bulk of the energy supply
for biosynthesis is obtained from oxidation of
acetyl-CoA in the Krebs cycle. This creates a
125
junction at isocitrate in which the flux of carbon is
divided between ICL and isocitrate dehydrogenase
(ICDH) in the ratio of I : 2. The partition of flux is
regulated by the reversible inactivation of ICDH
[7,8] effected by phosphorylation/dephosphorylation of the enzyme [9--11]. The regulatory enzyme
is a bifunctional ICDH-kinase/phosphatase
[12,13] which responds to a large number of signals [14-16] and achieves a very precise balance
between the competing pathways [3]. This is essential for growth because ICL has a much lower
affinity for isocitrate (K m = 604 /~M) than does
ICDH (K m = 8 #M) [17] and flux through ICDH
must be restricted to allow the intracellular concentration of isocitrate to rise [18] to a level sufficient to sustain flux through ICL.
If both functions of the ICDH-kinase/phosphatase were selected simultaneously it would have
been possible for the whole system to evolve using
pathways similar to those now found in the evolved
organism. However, if the kinase and phosphatase
functions evolved sequentially the kinase must have
arisen first because there could have been pressure
to select the phosphatase until the kinase was
available to generate ICDH-phosphate. These considerations pose a problem. The equilibrium of
ICDH-kinase favours phosphorylation [13] and the
primaeval organism would have to grow using
only the minimal ICDH activity available at or
near the equilibrium constant of ICDH-kinase.
How could this have been achieved?
3. THE GLYOXYLATE OXIDATION CYCLE
- - AN HYPOTHESIS
The glyoxylate bypass is a cycle which starts
with oxaloacetate and acetyl-CoA, regenerates the
oxaloacetate and produces a molecule of glyoxylate (Fig. 2). In short, the bypass oxidises acetylCoA to glyoxylate. The Krebs cycle oxidises
acetyl-CoA to CO 2 and generates reduced nucleotides used for ATP synthesis (FADH2,
NADH,H ÷) as well as for reductive biosynthesis
(NADPH,H+). If flux through the Krebs cycle
was minimised by ICDH phosphorylation, how
could the preimaeval organism make up the shortfall in energy supply? One solution could have
been to oxidise glyoxylate using three enzymes
(malate synthase, malic enzyme and pyruvate
dehydrogenase) found in the modern organism
which can be used in a cyclic process to oxidise
glyoxylate to CO 2 (Fig. 3). In this cycle glyoxylate
and acetyl-CoA are combined, two molecules of
CO 2 are evolved and acetyl-CoA is regenerated.
The combination of this glyoxylate-oxidising cycle
and the glyoxylate bypass provides the precursors
and reducing power required for biosynthesis with
minimal flux through ICDH (Fig. 3). The glyoxylate bypass together with either the glyoxylate
oxidising cycle or the Krebs cycle yields the same
end products both in terms of precursors and total
reducing power. The ratio of N A D H / N A D P H is,
however, different. On the other hand the modern
organism possesses two malic enzymes [19,20] one
of which is linked to NAD and the other to
NADP. If both of these enzymes were available to
the primaeval organism the N A D H / N A D P H ratio
would have been adjusted to the requirements of
the evolving organism. The proposition is, therefore, that evolution of the modern organism could
have involved a stage where ICDH-kinase inactivated a large fraction of the ICDH activity and
the glyoxylate-oxidising cycle generated reducing
power for biosynthesis. Even if the ICDH-phosphatase had been available at this time, the alternative oxidative cycle could still have been necessary until the precise control of kinase/phosphatase by allosteric effectors had been selected. If
the amount of ICDH cativity was insufficient to
sustain the flux to oxoglutarate shown in Fig. 3,
the evolving organism would simply have grown
more slowly until further regulatory improvement
in the system was selected. In this scheme ICDH is
relegated to a biosynthetic function as proposed
by Gest [21] as a stage in evolution of the Krebs
cycle. It is even possible that the glyoxylate-oxidising cycle antedated the Krebs cycle. The primaeval
organism could have used the glyoxylate-oxidising
cycle by virtue of an intrinsically low ICDH activity not requiring modulation by ICDH-kinase/
phosphatase. In this scenario the evolution of the
Krebs cycle would have required an increase in
ICDH activity which in turn would have exerted
the pressure to select a means to control the extra
activity to permit ICL to operate when acetate was
126
0.72
OAA
__/_
12, 1
pG
~
~1~
0.58
rP
G6P
0.4f
/
c°2
/
~~pAyiP
_PAYP
I L0
.I1 /
~CIT
~L Gly°xylateF ~
/I oxldlslng~CO
F~l~9~ cycle ~NA~H /
~ ~ Ac.CoA
FUM
"~ 1.13
FADH2~-'~
GI y
O
×
~
I ~
Acetate
..... '--F~ Input
c
D
H
SUC'~
0.4~5
C02
Fig. 3. Proposed pathway for growthon acetate with minimalparticipationof ICDH. Abbrviationsas in Figs. 1 and 2.
the only available carbon source. Evolutionary
arguments often seem plausible in more than one
way but, whatever the order of events, it seems at
least possible that the glyoxylate-oxidising cycle
had some function in the evolution of aerobic
metabolism in the organism now known as E. coli.
4. THE GLYOXYLATE-OXIDATION CYCLE
IN THE MODERN ORGANISM
Finally, what is the position of the glyoxylateoxidising cycle in the modern organism? Looking
at Fig. 3 there seems to be no reason why all three
cycles should not operate today because E. coli
possesses the two enzymes required to bridge the
gap between oxoglutarate and succinate. The relative fluxes through each would reflect the interac-
tion of controls imposed on each system. The
possible role of the glyoxylate-oxidising cycle in
evolved organisms is supported by recent work by
La Porte et al. [22] who isolated mutants lacking
ICDH-kinase which could not grow on acetate.
They then isolated pseudorevertants which had
regained the ability to grow on acetate. One class
of these had only 2.5% of the ICDH activity
required to sustain growth on acetate by means of
the glyoxylate bypass and the Krebs cycle (Fig. 2).
As these strains grew on acetate as quickly as the
parent strain it follows that they must possess
some mechanism, other than the Krebs cycle, which
oxidises acetate to provide reducing power for
biosynthesis. It seems at least possible that the
glyoxylate-oxidising cycle (Fig. 3) fulfills this function in this evolved organism. Certainly, if it does
not, some other very similar metabolic pathway
must by operating.
127
5. C O N C L U S I O N
It is p r o p o s e d that e v o l u t i o n of g r o w t h o n
acetate could have involved a glyoxylate-oxidising
cycle a n d that this s y s t e m m a y even have s o m e
f u n c t i o n in the m o d e r n o r g a n i s m .
ACKNOWLEDGEMENTS
T h i s w o r k has b e e n s u p p o r t e d b y g r a n t s f r o m
the S E R C a n d has b e n e f i t t e d greatly f r o m discussions with Drs. M a n s i E1-Mansi, L.M. Fixter, I.D.
H a m i l t o n , I.S. H u n t e r a n d Prof. C.A. F e w s o n .
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