The Glyoxylate Shunt, 60 Years On: Annual Review of Microbiology

MI72CH15_Welch ARI 11 August 2018 7:53
Annual Review of Microbiology
The Glyoxylate Shunt,

60 Years On
Stephen K. Dolan and Martin Welch
Annu. Rev. Microbiol. 2018.72:309-330. Downloaded from www.annualreviews.org
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW,

United Kingdom; email: skd41@cam.ac.uk, mw240@cam.ac.uk
Access provided by 178.255.168.83 on 03/13/20. For personal use only.
Annu. Rev. Microbiol. 2018. 72:309–30 Keywords

The Annual Review of Microbiology is online at glyoxylate shunt, isocitrate lyase, malate synthase, TCA cycle, isocitrate,
micro.annualreviews.org
acetate metabolism
https://doi.org/10.1146/annurev-micro-090817-
062257 Abstract
Copyright c 2018 by Annual Reviews. 2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate
All rights reserved
shunt (and coincidentally, also the 80th anniversary of his discovery of the
citric acid cycle). Sixty years on, we have witnessed substantial developments
in our understanding of how flux is partitioned between the glyoxylate shunt
and the oxidative decarboxylation steps of the citric acid cycle. The last
decade has shown us that the beautifully elegant textbook mechanism that
regulates carbon flux through the shunt in E. coli is an oversimplification of
the situation in many other bacteria. The aim of this review is to assess how
this new knowledge is impacting our understanding of flux control at the
TCA cycle/glyoxylate shunt branch point in a wider range of genera, and
to summarize recent findings implicating a role for the glyoxylate shunt in
cellular functions other than metabolism.
309
Contents
BACKGROUND AND A BRIEF HISTORY OF THE GLYOXYLATE SHUNT . . 310
THE TEXTBOOK MECHANISM OF TCA CYCLE–GLYOXYLATE SHUNT
FLUX PARTITIONING IN E. COLI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
CONTROL OF AceK KINASE/PHOSPHATASE ACTIVITY IN E. COLI. . . . . . . . . 312
STRUCTURAL INSIGHTS INTO THE MECHANISM OF E. COLI AceK . . . . . . . 313
THE aceBAK OPERON: GENETIC CONTEXT AND TRANSCRIPTIONAL
CONTROL OF THE ACETATE OPERON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
MICROBIAL PATCHWORK—INSIGHTS FROM 20 YEARS
OF BACTERIAL GENOME SEQUENCING. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
IS THE E. COLI REGULATORY MODEL REPRESENTATIVE OF OTHER
BACTERIA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
TRANSCRIPTIONAL REGULATION OF THE GLYOXYLATE SHUNT IN

DIFFERENT BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

POSTTRANSLATIONAL REGULATION OF THE GLYOXYLATE SHUNT
ENZYMES—BEYOND ISOCITRATE DEHYDROGENASE
PHOSPHORYLATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
THE GLYOXYLATE SHUNT AS A DRUG TARGET . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
FLUX THROUGH THE GLYOXYLATE SHUNT AFFECTS BACTERIAL
VIRULENCE FACTOR PRODUCTION, PERSISTENCE, AND
ANTIBIOTIC RESISTANCE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
METABOLON FORMATION AND SUBSTRATE CHANNELING . . . . . . . . . . . . . 325
METABOLIC ENGINEERING—APPLICATIONS OF THE
GLYOXYLATE SHUNT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
PARTING COMMENTS—LOOKING TO THE FUTURE . . . . . . . . . . . . . . . . . . . . . . 326
BACKGROUND AND A BRIEF HISTORY OF THE GLYOXYLATE SHUNT

The citric acid or tricarboxylic acid (TCA) cycle, elucidated by Krebs and Johnson in 1937, is
widely acknowledged as the central metabolic hub of the cell. The TCA cycle takes in carbon (as
acetyl-CoA) and oxidizes the two carbon atoms in each incoming acetyl moiety to two molecules
of CO2 . In doing so, it releases four pairs of electrons for energy production (38, 74). A special
problem arises when organisms are required to grow on substrates such as acetate, fatty acids,
or ketogenic amino acids as a sole carbon source. These substrates are degraded exclusively to
acetyl moieties, so in the absence of a mechanism to conserve some of the contained carbon for
gluconeogenesis (and hence, biomass production), no growth should be possible. However, many
bacteria do readily grow on these substrates, suggesting that they have evolved a mechanism to
redirect carbon away from the oxidative steps of the TCA cycle for gluconeogenesis. Indeed, when
Kornberg incubated suspensions of bacteria with 14 C-labeled acetate, he noticed that intermediates
of the TCA cycle acquired labeled carbon not only from the turning on of the TCA cycle, but
also from an ancillary pathway that must feed acetate in through a second point of entry (33,
37). Such an ancillary sequence had also been shown using cell-free extracts of acetate-grown
bacteria and resulted from the combined activity of two previously characterized enzymes. One of
these condensed acetyl-CoA with glyoxylate to form malate, and the other provided the glyoxylate
needed for this reaction by cleaving isocitrate (2, 36, 71). Together, these two steps enabled the
310 Dolan · Welch

O
CH3 C S-CoA
Acetyl-CoA
Gluconeogenesis Oxaloacetate
O C COO– CH2 COO–
CH2 COO– HO C COO– Citrate
CH2 COO–
COO– O
HO CH CH2 COO–
CH3 C S-CoA
Malate CH2 H C COO– Isocitrate
Acetyl-CoA ICL
COO– HO C COO–
H
O C H
MS IDH
COO–
ICD
Glyoxylate
COO– CO2
Fumarate CH CH2 COO–

HC CH2 α-Ketoglutarate
COO– C COO–
O
CH2 COO–
CH2 COO–
CH2
CH2
COO–
C S-CoA
Succinate
O
Succinyl-CoA
CO2
Figure 1
Overview of the glyoxylate shunt (red arrows) and its interaction with the TCA cycle. The oxidative
decarboxylation steps of the TCA cycle that are bypassed by the glyoxylate shunt are shown in gray. Acetyl-
CoA input moieties are highlighted in tan, and the enzymes discussed in the text are shown in orange (MS),
green (ICL), blue, and purple (ICD and IDH). Note that ICD and IDH are not always present together in
any given organism; many organisms encode either ICD or IDH. For clarity, the ICD kinase/phosphatase,
AceK, is not shown. Abbreviations: ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; IDH, monomeric
ICD; MS, malate synthase.
organism to bypass the oxidative decarboxylation steps of the TCA cycle, thereby conserving
carbon skeletons for biomass generation. For this reason, the sequence, first discovered in 1957
by Kornberg & Krebs (35), was termed the glyoxylate bypass. The net effect of this bypass is
the formation of one four-carbon dicarboxylic acid (malate, a gluconeogenic precursor) from two
molecules of acetyl-CoA input (15, 32).
As shown in Figure 1, the glyoxylate bypass siphons carbon away from the main sequence of
the TCA cycle at isocitrate, immediately before the commencement of oxidative decarboxylation.
The glyoxylate shunt generates succinate and malate, which are subsequently oxidized to yield
fumarate and oxaloacetate (respectively), so it also provides some energy to the cell. However, this
is not an all-or-none switch between competing pathways; even during growth on acetate, flux is
partitioned between the main sequence of TCA cycle reactions and the glyoxylate shunt, thereby
maintaining a balance of energy and gluconeogenic precursor production. Recent developments
in our understanding of the enzymology and genetics underpinning this flux partitioning are the
main focus of this review. Before diving into those recent developments though, it is worth pausing
www.annualreviews.org • The Glyoxylate Shunt 311

to review our current understanding of how flux through this important metabolic branch point
is regulated in Escherichia coli—the best understood system.
THE TEXTBOOK MECHANISM OF TCA CYCLE–GLYOXYLATE

SHUNT FLUX PARTITIONING IN E. COLI
The glyoxylate shunt was first discovered in Pseudomonas fluorescens KB1. However, most of what
we now know about its enzymology is based on work carried out in E. coli (34, 35, 43). The
first dedicated enzyme in the pathway, isocitrate lyase (ICL; encoded by aceA), is a tetramer that
catalyzes the aldol cleavage of isocitrate to yield succinate (an intermediate of the TCA cycle)
and glyoxylate. Following this, malate synthase (MS; encoded by aceB in E. coli) catalyzes the
Claisen-like condensation of glyoxylate with acetyl-CoA to yield malate and CoA (Figure 1). The
net result is an anaplerotic reaction in which two molecules of acetyl-CoA yield one molecule of
malate. The latter can then be redirected away from the main sequence of TCA cycle reactions
to generate glucose (and, hence, cellular biomass) through gluconeogenesis.
In E. coli, NADP-dependent isocitrate dehydrogenase (ICD) catalyzes the conversion of iso-

citrate to α-ketoglutarate. When bacteria are grown on acetate, ICD competes with ICL for their
common substrate, isocitrate. ICL has a much lower affinity for isocitrate [KM = 63–604 μM in
vitro, depending on the anion concentration and likely greater than this in vivo where inhibitory
anion concentrations are high (44, 49)] than ICD (KM = 8 μM), so flux through the latter must be
restricted to allow the intracellular concentration of isocitrate to rise to a level sufficient to sustain
flux through the glyoxylate shunt (42). ICD from E. coli has traditionally been viewed as being in-
sensitive to most allosteric effectors, although in 2007 Ogawa et al. (63) reported that the enzyme is
inhibited by phosphoenolpyruvate (PEP). Instead, ICD activity is primarily controlled by an ICD
kinase/phosphatase called AceK (23, 42). AceK is a bifunctional enzyme that specifically catalyzes
the reversible phosphorylation and dephosphorylation of ICD; in fact, this was the first example
of phosphorylation identified in any prokaryote. ICD is phosphorylated by AceK on Ser113 in
the active site of the dehydrogenase. Insertion of a negatively charged phosphate group into the
active site of ICD causes electrostatic repulsion of the isocitrate substrate, effectively inactivating
the enzyme. This inactivation is reversed by the phosphatase activity of AceK. The differential
kinase/phosphatase activity of AceK determines the extent of ICD phosphorylation, which in turn
controls the balance of the carbon flux between the TCA cycle and the glyoxylate shunt in E. coli.
When bacteria are grown on acetate, about 75% of ICD is inactivated by phosphorylation (43, 78).
CONTROL OF AceK KINASE/PHOSPHATASE ACTIVITY IN E. COLI

The AceK kinase/phosphatase activity is allosterically regulated by several key metabolites in
vitro, although the precise mechanism(s) by which they accomplish this is still not fully clear. AceK
comprises two domains: a regulatory domain and a catalytic domain. The latter carries out both the
kinase and the phosphatase activities. AMP, 3-phosphoglycerate, and pyruvate are thought to bind
to the regulatory domain of AceK directly, stimulating the phosphatase activity and concomitantly
inhibiting the kinase (57). Because AceK is likely saturated in vivo with its substrate, ICD, such dual
effect regulators can give rise to an interesting mechanism of sensitivity amplification known as
zero-order ultrasensitivity (41). Although there are no known regulators that specifically activate
the kinase activity of AceK without affecting the phosphatase, there are some specific kinase
inhibitors, fructose-6-phosphate and glyoxylate. One particularly interesting regulator is ATP. As
the phosphate donor, ATP is an absolute prerequisite for the kinase activity of AceK, but at the
same time, it is also a potent activator of the AceK phosphatase, too (57, 62, 86). This confers an
312 Dolan · Welch

ATPase property on AceK, presumably reflecting the metabolic cost that accompanies sensitive
regulation. Isocitrate and NADPH also appear to regulate ICD phosphorylation, although they do
so indirectly by altering the accessibility of Ser113 on ICD (through steric competition in the case
of isocitrate or via global conformational change in the case of NADPH) (57). Collectively then,
we can view these allosteric regulators as shifting two types of equilibria in the cell: the equilibrium
between the kinase-active or phosphatase-active conformers of AceK, and the equilibrium between
different conformers (active site open or active site closed) of the substrate, ICD. However, it is
difficult to discern any obvious pattern as to why certain metabolites stimulate kinase versus
phosphatase activity in E. coli AceK. It has been suggested that the key regulator is likely to be
the substrate, isocitrate (40). When the pool of isocitrate is lowered, more ICD will become
phosphorylated, and consequently more isocitrate will be directed through the glyoxylate shunt.
Interestingly, more recent work has shown that in addition to allosterically inhibiting ICD, PEP
also uncompetitively inhibits ICL (63). Thus, as more isocitrate becomes available as a consequence
of PEP-dependent inhibition of ICD, PEP also begins to bind to the ICL-isocitrate enzyme-
substrate complex, slowing down flux through the glyoxylate shunt. This generates an additional
layer of feedback control in which flux through the two competing pathways become differentially
autolimited in response to changing PEP concentrations. The physiological significance of this is
that PEP may be an indicator of the fate of oxaloacetate in the cell. Oxaloacetate can be converted
to citrate through the action of citrate synthase, or it can be redirected toward gluconeogenesis (and
PEP production) as a consequence of PEP carboxykinase activity. Increased flux through citrate
synthase would lower the PEP concentration and thereby relieve the uncompetitive inhibition of
ICL, restoring oxaloacetate production via the glyoxylate shunt. PEP-dependent control of flux
through ICL also links the glyoxylate shunt with carbohydrate uptake via the phosphotransferase
system; PEP is the ultimate phosphorous donor for most phosphotransferase sugar uptake systems,
so when carbohydrate uptake is high, PEP levels would be expected to fall accordingly.
STRUCTURAL INSIGHTS INTO THE MECHANISM OF E. COLI AceK

Remarkably, and in spite of its function as a central metabolic integrator controlling flux parti-
tioning through a vital metabolic branch point, the enzymology of AceK remains relatively poorly
investigated. As with any field of human endeavor, interest in this topic seems to have waxed
and waned with the retirement or shifting interests of the researchers involved. However, a key
breakthrough in our understanding of AceK function has been the long-overdue resolution of its
X-ray crystal structure. In a tour de force, in 2010 Zheng & Jia (86) presented the structures of free
and ICD-bound AceK. AceK contains two distinct domains, an N-terminal regulatory domain
with a unique fold, and a C-terminal catalytic domain with similarity to certain eukaryotic protein
kinases. Zheng and Jia obtained crystal structures in the presence and absence of a known allosteric
regulator, AMP. In the AMP-bound form, the ligand was located in a pocket between the regula-
tory and catalytic domains. AMP binding elicits a set of local conformational changes that cascade
down to the ATP-binding site some 25 Å away. In essence, in the kinase inactive/phosphatase
active AMP-bound structure, the β3αC loop blocks the active site and prevents ATP binding. In
contrast, in the absence of AMP (i.e., in the kinase active/phosphatase inactive configuration), the
β3αC loop moves away from the active site, allowing ATP to bind. Presumably, other regulators
(binding elsewhere on the regulatory domain of AceK) elicit comparable conformational changes.
As might be expected, deletions in the β3αC loop reduce the phosphatase activity of AceK and
increase its kinase activity (81). Intriguingly, the same deletions also increase the affinity of AceK
for ICD. Given that the β3αC loop forms part of the substrate-binding cleft, it is not clear why
this should be. The other major component of the ICD-binding cleft is a loop structure (residues

484–510) denoted the substrate-recognition loop, or SRL. The SRL projects away from the main
body of AceK, penetrating deeply into the active site of ICD. Apo-ICD is a dimer in which the
phosphorylatable serine residue, Ser113, is hidden deep within a pocket and points away from the
surface. However, upon binding AceK (in the stoichiometry of one monomer of AceK per pro-
tomer of ICD dimer) the ICD undergoes a conformational change resulting in Ser113 adopting
an outward-facing, more exposed pose. This appears to be driven, at least in part, by penetration
of the SRL deep into the ICD active site. This twists and deforms the active site, yielding the
observed inversion of Ser113 configuration, priming it for subsequent phosphotransfer. Intimate
contacts made between the side chains of the SRL and the ICD active site confer specificity
in this interaction. In this regard, it is interesting to note that these interactions are conserved
among ICD orthologs in gram-negative bacteria but are disrupted or absent in ICD proteins from
gram-positive bacterial species. Consistent with this, E. coli AceK can phosphorylate ICD from
Burkholderia pseudomallei (80) but fails to efficiently phosphorylate ICD from Bacillus subtilis (69).
This may explain why AceK (and hence, AceK-dependent regulation of flux through the glyoxylate
shunt) is a feature associated only with gram-negative bacteria (80). Atomic resolution structures
of E. coli ICL and MS have also been described in the literature (8, 72).
THE aceBAK OPERON: GENETIC CONTEXT AND TRANSCRIPTIONAL

CONTROL OF THE ACETATE OPERON
In E. coli, the open reading frames (ORFs) encoding MS (aceB), ICL (aceA), and ICD kinase/
phosphatase (aceK) form a tricistronic operon (aceBAK) located at 90.85 min on the E. coli K-
12 linkage map (13) (Figure 2). Expression of the aceBAK operon is primarily controlled by IclR
(isocitrate lyase regulator), which binds tightly to the operator region, repressing expression of the
gene cluster. However, during growth on acetate or fatty acids, IclR repression is relieved and the
operon is transcribed. The nature of the inducer molecule(s) is not yet clear, although glyoxylate
and pyruvate elicit intriguing antagonistic changes in IclR in vitro. For example, glyoxylate was
shown to prevent IclR binding to the aceBAK operator (thereby stimulating expression of the
operon) by stabilizing the inactive dimeric state of the protein, whereas pyruvate increased the
binding of IclR to the aceBAK promoter by stabilizing the active, tetrameric form of the protein
(14, 15, 27, 48). Interestingly, these effectors appear to bind to the same site on IclR (48). PEP has
also been shown to prevent IclR binding to the aceBAK promoter in vitro (14). IclR also negatively
regulates its own expression, although this repression appears to be ligand independent. However,
expression of iclR is also under positive control by another transcriptional regulator, FadR, which
additionally coordinates the expression of genes involved in fatty acid metabolism. FadR represses
the expression of genes involved in β-oxidation and concomitantly activates a subset of genes
involved in the biosynthesis of fatty acids. Mutants in fadR show increased aceBAK expression
(because iclR is no longer transcribed) and increased expression of the β-oxidation enzymes.
The IclR/FadR regulatory pair therefore nicely coordinate the breakdown of fatty acids with the
generation of gluconeogenic precursors via glyoxylate shunt activity (27).
MICROBIAL PATCHWORK—INSIGHTS FROM 20 YEARS

OF BACTERIAL GENOME SEQUENCING
The dimeric AceK-sensitive ICD enzyme from E. coli is not the only type of microbial ICD. From
the late 1960s onward, it was increasingly clear that some organisms contain a monomeric ICD
(hereafter, IDH). However, it was not until 1993 that Ishii et al. (31) reported the first sequence
of an IDH enzyme. They found that the psychrophilic Vibrio sp. strain ABE-1 (now known as
314 Dolan · Welch

a
IclR IclR aceB aceA aceK yjaC iclR
MS ICL AceK IclR
b Escherichia coli
Glyoxylate shunt TCA cycle
Isocitrate
ATP ADP Inactive

KM= 8 µM
KM= 604 µM P
ICL
ICD ICD
ICD
AceK
Glyoxylate α-Ketoglutarate
3-phosphoglycerate,
pyruvate, citrate,
AMP, ADP, isocitrate,
MS oxaloacetate, PEP,
α-ketoglutarate, NADP+
c Mycobacterium spp.
Glyoxylate shunt TCA cycle
Isocitrate
KM= 20 µM
KM= 145 µM
ICD
ICL
MS
Figure 2
Organization of the Escherichia coli aceBAK operon and comparison of branch point regulation in E. coli and
Mycobacterium spp. (a) The aceBAK operon and its regulation by IclR (dotted arrow). yjaC is an
uncharacterized gene. (b) Enzymology of the glyoxylate shunt–TCA cycle branch point in E. coli,
highlighting the importance of AceK-mediated control of flux. Several metabolites are known to affect the
kinase/phosphatase activity of this enzyme (red, dotted, bar-headed line). (c) Enzymology of the glyoxylate
shunt–TCA cycle branch point in Mycobacterium spp. Note that the product of the ICL-catalyzed reaction,
glyoxylate, cross-activates ICD, setting up a rheostatic control mechanism. The KM values in this panel are
taken from Reference 60. Abbreviations: ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; IDH,
monomeric ICD; MS, malate synthase.
Colwellia maris) encodes both the dimeric (ICD) and monomeric (IDH) isozymes. Sequencing of
the C. maris icd and idh genes revealed that although icd was very similar to the E. coli gene, the
idh sequence was (at that time) unique. Furthermore, the IDH isozyme is almost certainly AceK
insensitive. More recent genome sequencing efforts have revealed that the icd and idh genes are
widespread among bacterial genera. Some species, such as the industrially important fermenter

Corynebacterium glutamicum contain only idh. Others, like E. coli, contain only icd. Yet others,
such as the intracellular pathogen Mycobacterium tuberculosis contain both icd (Mtb ICD-1) and idh
(Mtb ICD-2) but lack aceK. A small number of organisms also appear to encode more than one
ICL isoform. For example, M. tuberculosis contains an E. coli–like ICL isoform (ICL-1, encoded
by the icl gene, Rv0467) that plays an important role in pathogenicity and intracellular survival
in macrophages (53), and also a less-characterized isoform (denoted AceA or ICL-2, encoded by
Rv1915/Rv1916). Both isozymes are jointly required for optimal virulence and fatty acid catabolism
in M. tuberculosis (59). One possible explanation for the apparent redundancy here is that ICL-1
has both ICL and 2-methylisocitrate lyase activity. The latter is associated with the methylcitrate
cycle and is surprising because structural and enzymatic work previously suggested that these are
two distinct enzyme families with little or no overlap in function (25). It is also worth noting that
in many cases, the ORFs encoding the acetate catabolism genes (aceB, aceA, and aceK in E. coli)
are not linked, perhaps indicative of more complex genetic regulation. A snapshot of the diversity
of genes present in a selection of bacteria is shown in Table 1. Although certain catalytic and
regulatory motifs are discernible, it is clear that the enzymology of the branch point enzymes
must be more complicated than can be inferred by extrapolation from the E. coli model.
ICD plays a key role in the cell as its principal supplier of reducing equivalents (such as NADPH)
for anabolism and for combatting oxidative stress. Indeed, during growth on acetate, ICD provides
90% of the NADPH necessary for biosynthesis. However, not all ICDs utilize NADP+ ; some
exhibit a preference for NAD+ , although bacteria encoding exclusively NAD-dependent ICDs
(a) lack ICL, (b) are incapable of growth on acetate, and (c) lack either a respiratory chain or
a complete Krebs cycle (18). Why some organisms encode more than one NADP-dependent
isocitrate dehydrogenase (e.g., ICD and IDH) is not quite clear (87). One possibility is that this is
a belt-and-braces strategy to ensure that the organism never runs short of NADPH for combatting
oxidative stress, especially during infection scenarios.
Cyanobacteria are a large group of oxygenic chlorophototrophic bacteria with highly diverse
metabolic capabilities, but the occurrence of the glyoxylate cycle in these organisms has remained
controversial (83). However, a detailed genomic analysis aimed at cataloging metabolic traits of
cyanobacteria reported that two Cyanothece strains (PCC 7424 and PCC 7822) contain an operon
encoding ICL and MS (4). A similar operon has also been identified in Chlorogloeopsis fritschii
(strain PCC 9212), and the enzymes encoding ICL and MS have been biochemically validated
(84).
IS THE E. COLI REGULATORY MODEL REPRESENTATIVE OF OTHER

BACTERIA?
The notion that flux partitioning between the TCA cycle and the glyoxylate shunt is primarily
controlled by reversible phosphorylation of one of the branch point enzymes lies at the heart
of the E. coli regulatory paradigm. But is this regulatory model also applicable to other species?
A full biochemical characterization encompassing analysis of the enzymology, flux partitioning,
posttranslational modifications, and transcriptional regulation has been carried out in detail only
for M. tuberculosis. Based on these analyses, the mechanism of flux control appears to be totally
different from the canonical E. coli model of flux partitioning.
Like E. coli, mycobacteria appear to exclusively use the TCA cycle during growth on glucose but
switch to partitioning of flux between the TCA cycle and glyoxylate shunt during growth on acetate
(60). However, Mycobacterium spp. do not encode an AceK homolog, and it seems unlikely that any
other enzyme plays a comparable role. Instead, an AceK-independent, rheostat-like mechanism has
been proposed to control flux bifurcation in this organism (60) (Figure 2). M. tuberculosis encodes
316 Dolan · Welch

MI72CH15_Welch
Table 1 Distribution of the glyoxylate shunt—TCA cycle branch point enzymes in a selection of bacterial generaa
Species ICL-1 ICL-2 AceK IDH ICD GlcB Observations
ARI
Staphylococcus aureus ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ citC ∗∗∗ NA

(NWMN_1587)
Haemophilus influenza ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
Listeria monocytogenes ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ citC (lmo1566) ∗∗∗ NA
11 August 2018
Streptococcus pneumoniae ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗
7:53
Helicobacter pylori HP0027 NA

Treponema pallidum ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
Borellia burgdorferi ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
Clostridium difficile ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ CD630_08340 ∗∗∗ NA
Mycoplasma pneumoniae ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
Rickettsia rickettsii ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ RrIowa_0425 ∗∗∗ NA
Chlamydia trachomatis ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ No shunt, no TCA
enzyme
Thermotoga maritima ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ TM1148 ∗∗∗ NA
Rhodospirillum rubrum ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ Rru_A0356 ∗∗∗ NA
Nitrosomonas eutropha ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ NE1730 ∗∗∗ NA
Campylobacter jejuni ∗∗∗ ∗∗∗ ∗∗∗ Cj0531 ∗∗∗ ∗∗∗ NA
Francisella tularensis ∗∗∗ ∗∗∗ ∗∗∗ FTN_1434 ∗∗∗ ∗∗∗ NA
Neisseria meningitidis ∗∗∗ ∗∗∗ ∗∗∗ NMB0920 ∗∗∗ ∗∗∗ NA
Acinetobacter baumanii A1S_1008 ∗∗∗ ∗∗∗ A1S_2477 A1S_2475 A1S_1601 NA
Aeromonas hydrophila AI20_05255 ∗∗∗ AI20_04295 ∗∗∗ AI20_11960 AI20_05260 aceB (glcB) and aceA are
adjacent (in an
www.annualreviews.org • The Glyoxylate Shunt

operon?), aceK is
unlinked
317
Azotobacter vinelandii Avin_28420 ∗∗∗ ∗∗∗ Avin_28310 ∗∗∗ Avin_43290, Avin_43290 part of a
Avin_05920 glycollate degradation
operon? Avin_05920
not part of aceBAK-like
operon
(Continued)
Table 1 (Continued)
Species ICL-1 ICL-2 AceK IDH ICD GlcB Observations
MI72CH15_Welch
Bacillus anthracis BAS1052 ∗∗∗ ∗∗∗ ∗∗∗ BAS4487 BAS1051 aceBA operonic
Bordetella pertussis BP2085 ∗∗∗ ∗∗∗ ∗∗∗ BP2488 BP3680 NA
ARI
Corynebacterium Cgl2331 ∗∗∗ ∗∗∗ Cgl0664 ∗∗∗ Cgl2329 aceA and aceB (glcB)
glutamicum divergently transcribed
318
Pectobacterium ECA3990 ∗∗∗ ECA3989 ∗∗∗ ECA2439 ECA3991 aceBAK operonic
atrosepticum
Dolan
Burkholderia cepacia GEM_4139 GEM_1393 GEM_0511 GEM_0907 GEM_0906 GEM_5590 icd and idh adjacent and
·
11 August 2018
in same orientation
Hahella chejuensis HCH_02323 ∗∗∗ ∗∗∗ HCH_03212 HCH_02336 HCH_1653 NA
Welch
∗∗∗ ∗∗∗
7:53
Klebsiella pneumoniae KPNIH24_01265 KPNIH24_01270 KPNIH24_18460 KPNIH24_01260 aceBAK operonic

Pseudomonas aeruginosa PA2634 ∗∗∗ PA1376 PA2624 PA2623 PA0482 NA
Proteus mirabilis PMI2763 ∗∗∗ PMI2762 ∗∗∗ PMI0891 PMI2764 aceBAK operonic
Ralstonia solanacearum RSc1358 ∗∗∗ RSc0278 ∗∗∗ RSc2490 ∗∗∗ Has aceA (ICL) but lacks
an aceB (glcB) homolog
Mycobacterium RV0467 RV1915, ∗∗∗ RV0066c RV3339c RV1837c NA
tuberculosis RV1916 (ICD-2) (ICD-1)
Rhizobium meliloti SMc00768 ∗∗∗ ∗∗∗ SMc00480 SMc02581 ICD might be closer
to tartrate
dehydrogenase?
Stenotrophomonas Smlt0232 ∗∗∗ Smlt4268 Smlt4273 Smlt0982 Smlt0231 aceBA operonic, ICD
maltophilia only 35% similar to E.
coli ICD
Vibrio cholerae VC0763 ∗∗∗ ∗∗∗ VC1141 ∗∗∗ VC0734 Uses IDH instead of
ICD, so no need for
aceK
Xanthomonas campestris XCC0238 ∗∗∗ XCC3780 XCC3782 XCC0967 XCC0237 XCC0237 has only weak
similarity with GlcB,
but still in operon with
aceA
Yersinia pestis YpAngola_A0640 ∗∗∗ YpAngola_A3925 ∗∗∗ YpAngola_A2848 YpAngola_A0639 aceBA operonic
Yersinia pseudotuberculosis YPK_0365 ∗∗∗ YPK_0366 YPK_1723 YPK_0364 aceBAK are operonic
Abbreviations: ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; IDH, monomeric ICD; NA, none available.
a
Note that for each species, results for only a single representative genome sequence (strain) are shown. In a small number of species, the presence or absence of a specific gene was strain
dependent (e.g., the ICD coding sequence in S. maltophilia). The table was constructed by BLAST searching the genome sequence of each selected strain with the protein sequence of ICL, ICD,
IDH, AceK, or GlcB from P. aeruginosa or the ICL-2 sequence from M. tuberculosis. Where no homolog was identified, this is indicated by ∗ ∗ ∗ . For each species, the gene identifiers
corresponding to hits displaying the highest sequence similarity (E > 10−5 ) with the query sequence are indicated.
both ICD (ICD-1) and IDH (ICD-2) homologs, although only the IDH homolog appears to be
important for TCA cycle flux under physiological conditions. Unlike the situation in E. coli, where
the KM of ICL for isocitrate is approximately 100 times the KM of ICD for the same substrate, in
mycobacterial species the ICL:IDH KM differential is lower (145 μM:20 μM, different by a factor
of just 7). In the flux rheostat model, the activity of IDH is stimulated by glyoxylate, which is ideally
suited as a regulator because it is a unique product of the ICL-catalyzed cleavage of isocitrate to
glyoxylate and succinate. Thus, as the flux of carbon through ICL increases, this gives rise to an
increase in the corresponding steady-state concentration of glyoxylate. This, in turn, feeds back
to activate ICD, thereby decreasing flux through the glyoxylate shunt (60). Consistent with this,
and in stark contrast with the situation in E. coli, total ICD activity in mycobacterial cell extracts
increases in acetate-grown cells compared with glucose-grown cells. This development fills a
significant gap in our understanding of how flux partitioning can be potentially regulated. It also
opens up exciting possibilities regarding the regulatory arrangements in species that contain not
only ICD and IDH but also a functional AceK protein (e.g., Pseudomonas aeruginosa or Burkholderia
cepacia; Table 1).
TRANSCRIPTIONAL REGULATION OF THE GLYOXYLATE SHUNT IN

DIFFERENT BACTERIA
Another clear divergence from the E. coli regulatory paradigm (Figure 2) is seen at the tran-
scriptional level. As indicated in Table 1, the glyoxylate shunt–encoding genes are not always
maintained in a polycistronic operon. For example, the ICL-, MS-, and AceK-encoding genes are
distributed across the genome in monocistronic units in P. aeruginosa, M. tuberculosis, Mycobacterium
smegmatis, and B. cepacia.
One of the more interesting developments has come from an analysis of transcriptional regula-
tion in the industrially important gram-positive species Corynebacterium glutamicum. This organism
does not encode a functional homolog of IclR, the transcriptional repressor of the aceBAK operon
in E. coli. Instead, transcriptional control of glyoxylate shunt gene expression is mediated by the
regulator of acetate metabolism, RamB. Unusually, this protein was identified by biochemical
means rather than through genetics (in the first instance). DNA-affinity chromatography was
used to isolate proteins able to bind to the upstream region of the pta-ackA operon (encoding
phosphotransacetylase-acetate kinase and acetate-activating enzymes) in crude extracts of C. glu-
tamicum. A 53-kDa protein was identified and subsequently also shown to be able to bind to the
intergenic region between the ICL- and MS-encoding genes. In the absence of acetate, RamB
represses both aceA (ICL) and aceB (MS) gene expression and pta-ack expression (24). This facili-
tated the discovery of a ramB homolog in a related actinomycete, M. tuberculosis. Inspection of the
promoter region of the M. tuberculosis aceA gene revealed a 13-bp DNA element (CAAAATTTG-
CAAA) very similar to the RamB binding motif found in the aceA-aceB intergenic region of
C. glutamicum (24). Interestingly, this regulator very specifically repressed the synthesis of icl1 and
ramB itself and did not affect the expression of any other genes involved in acetate metabolism in
M. tuberculosis (56). The function of RamB in M. tuberculosis appears to be to repress icl1 expression
during growth on glucose. Subsequent work has shown that PrpR, a transcription factor involved
in regulation of the methylcitrate pathway in M. tuberculosis, acts as a repressor of ramB expression,
highlighting how these two distinct pathways are integrated in this organism (51) (Figure 3).
Another good example of the diverse strategies used to regulate glyoxylate shunt gene expres-
sion is seen in P. fluorescens. Here, the expression of aceA is regulated by RccR, a homolog of
the Entner-Doudoroff pathway regulator HexR. Like HexR, the DNA-binding activity of RccR is
modulated by the Entner-Doudoroff intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG).

MI72CH15_Welch
a RccR regulon in Pseudomonas spp. b RamB/PrpR regulon in Mycobacterium spp.

aceA
Glucose
ARI
ramB
glcB prpDC
KDPG
320
rccR RamB
GAP
CO2 Gap icl1
Dolan
KDPG
PrpR
·
PEP
11 August 2018
RccR RccR Propionate

PckA Pyruvate
Pyruvate
Welch
CO2 CO2
CO2 Propionyl-CoA
7:53
AceE F Acetyl-CoA
Acetyl-CoA Acetate
Acetate Citrate
Citrate 2-Methylcitrate Oxaloacetate
Oxaloacetate PrpC
NADH Isocitrate
NADH Isocitrate
PrpD Acetyl-CoA
Acetyl-CoA Malate NADPH
Malate NADPH 2-Methyl-cis-aconitate CO2
MS ICL1
MS CO2
ICL Glyoxylate α-Ketoglutarate
NADPH
NADPH CO2
CO2 2-Methylisocitrate Fumarate 2H
Fumarate Succinyl-CoA
Succinyl-CoA
ICL1
Succinate
2H Succinate
Pyruvate
Figure 3
Transcriptional regulation by RccR and RamB transcriptional regulation of glyoxylate shunt gene expression. (a) The RccR regulon. Binding of the Entner-Doudoroff
intermediate, KDPG, to RccR increases the affinity of this transcriptional regulator for the upstream regions of aceA and glcB, thereby repressing expression of the
glyoxylate shunt enzymes in the presence of glucose. RccR also regulates expression of genes involved in pyruvate metabolism (aceEF) and gluconeogenesis (pckA, gap).
RccR-regulated carbon transitions are marked in red. (b) The RamB and PrpR regulon in Mycobacterium spp. RamB represses icl expression and its own expression during
growth on glucose. PrpR, a transcription factor involved in regulation of the methylcitrate pathway of propionate degradation in M. tuberculosis, represses ramB
expression during growth on propionate. PrpR also activates the expression of icl, as this enzyme has methylisocitrate lyase activity and is required for propionate
degradation. RamB-regulated carbon transitions are marked in red. In both panels, dotted arrows indicate activation of transcription, while dotted, bar-headed lines
indicate repression of transcription. Abbreviations: ICL, isocitrate lyase; KDPG, 2-keto-3-deoxy-6-phosphogluconate; MS, malate synthase; PEP, phosphoenolpyruvate.
However, HexR and RccR control distinct aspects of primary metabolism in Pseudomonas; whereas
HexR controls the expression of genes involved in glucose catabolism and uptake, RccR regulates
pyruvate metabolism (aceEF, encoding subunits of pyruvate dehydrogenase), the glyoxylate shunt
genes [including aceA and glcB (encoding MS in this organism)] and gluconeogenesis (pckA, gap).
An unusual property of RccR is that it can switch between repression of pyruvate metabolism and
activation of the glyoxylate shunt/gluconeogenesis loci depending on the available carbon source.
This remarkable reciprocal regulation is brought about by the differential binding of RccR to two
distinct pseudopalindromic binding sites with the consensus sequence TGTAGT. . . . ACTACA.
These two sites have different linker lengths. Upstream of the glyoxylate shunt genes, the motif is
28 bp long, whereas upstream of the aceEF genes, the sequence is just 15 bp long. In the absence of
inducer (KDPG), RccR has a far higher affinity for the 15-bp site than for the 28-bp site, thereby
repressing expression of the aceEF genes. In contrast, in the presence of KDPG, the affinity of
RccR for the 28-bp site is increased, and binding to the 15-bp site is concomitantly decreased. This
way, P. fluorescens is able to coordinate gene expression in response to carbon source availability
(10, 17) (Figure 3).
POSTTRANSLATIONAL REGULATION OF THE GLYOXYLATE SHUNT

ENZYMES—BEYOND ISOCITRATE DEHYDROGENASE
PHOSPHORYLATION
Recent studies have shown that many enzymes involved in metabolism are acetylated on lysine
side chains and that this modification can reversibly alter the activity of those enzymes (79, 82).
Acetyl-CoA synthase (Acs) from Salmonella enterica was the first enzyme confirmed to be regu-
lated by lysine acetylation in bacteria (46, 73), but this modification has since been found to be
widespread among many species. For example, protein acetylation has been shown to directly
influence acetate metabolism in both S. enterica and E. coli. Indeed, around 90% of all central
metabolic enzymes exhibit this modification in S. enterica (79). Acetylation in the enterics appears
to be catalyzed by protein acetyltransferase (Pat), whereas deacetylation is catalyzed by an NAD-
dependent sirtuin-type enzyme called CobB. Consequently, mutants defective in CobB activity
display increased acetylation. In a wide-ranging study, Wang et al. (79) found that in S. enter-
ica, deletion of cobB leads to faster growth on glucose compared to wild type but slower growth
in the presence of citrate as a sole carbon source. In contrast, a pat mutant (with decreased
protein acetylation) exhibited the opposite growth phenotype. Subsequent 13 C-flux analyses re-
vealed that during growth on citrate, the pat mutant displayed increased flux (cf. the wild type)
through the glyoxylate shunt, whereas the cobB mutant exhibited greater flux through the gly-
colytic pathway (11, 79). To investigate this further, Wang et al. (79) purified ICL, AceK, and
a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (Gap), and examined the effect
of Pat/CobB-mediated acetylation/deacetylation on the activity of these enzymes in vitro. They
found that increased acetylation of ICL and AceK led to a decrease in the activity of these enzymes
(isocitrate cleavage and ICD phosphorylation, respectively). Given that the acetylation-mediated
decrease in AceK-dependent ICD phosphorylation resulted in increased ICD activity, these re-
sults indicate that reversible acetylation of ICL and AceK can modulate flux partitioning between
the TCA cycle and glyoxylate shunt. In contrast, increased acetylation of Gap led to increased
flux through the glycolytic pathway. However, it is worth noting that these effects of in vitro
acetylation of ICL and AceK could not be reproduced in a recent study, suggesting that more
work needs to be done in this area (16). Moreover, and unlike the substantial effect of phosphor-
ylation on ICD activity, acetylation appears to mediate more of a modulatory response, perhaps
fine-tuning fluxes in response to subtle changes in nutrient availability.

ICL has also been shown to be regulated by acetylation in E. coli, where 13 acetylation sites
were identified in the protein in vivo. Of these, 5 sites (K13, K34, K308, K326, and K331) showed
increased acetylation in a cobB mutant, although subsequent in vitro analyses revealed that CobB
exhibits a preference for deacetylation at only 2 of these sites (K13 and K308). However, this
deacetylation led to an increase (approximately 40%) in ICL activity. No evidence was found for
AceK acetylation in vivo though, and deletion of pat or cobB did not affect flux partitioning in an
aceK mutant (11).
Recent work by Bi et al. (7) has also uncovered a role for acetylation in modulating the activity
of ICL-1 in M. tuberculosis. ICL-1 was found to be acetylated on three lysine residues (K322, K331,
and K392). Interestingly though, the effect(s) of acetylation at each site were different. Using site-
directed mutagenesis, the team found that substitutions at position 392 generally enhanced ICL
activity, whereas substitutions at position 322 reduced ICL-1 activity. To investigate this further,
they used an elegant genetic system in which the codon for lysine (AAG) at positions 322 or 392 was
replaced by an amber (TAG) stop codon. The N -acetyllysyl-tRNA synthetase/tRNACUA pair of

Methanosarcina barkeri was then used to direct site-specific incorporation of N -acetyllysine to the
amber codon, resulting in uniform insertion of acetyllysine at the desired sites. This way, the team
confirmed the results from site-directed mutagenesis; furthermore, they observed that acetylation
of K322 (but not K392) led to diminished 2-methylisocitrate lyase activity (recall that ICL-1
has both ICL and 2-methylisocitrate lyase activity). In addition, acetylation of K392 affected the
abundance of ICL-1, apparently protecting the enzyme from turnover. These observations may
be significant, because the level of K392Ace ICL-1 markedly increased in M. tuberculosis cultures
grown in propionate or acetate (compared with cultures grown in glucose) (7).
Protein acetylation in M. tuberculosis is likely catalyzed by Rv2170, which encodes a protein
with lysine acetyltransferase activity, whereas deacetylation appears to be catalyzed by a sirtuin-like
protein, Rv1151c (NpdA). Rv2170 uses acetyl-CoA as an acetyl group donor to posttranslationally
modify proteins. In ICD, residues K30 and K129 are acetylated by Rv2170, and this leads to a
marginal reduction (approximately 30%) in ICD activity. Although this is nowhere near as potent
as phosphorylation in E. coli ICD, this observation does at least show that M. tuberculosis has some
potential to regulate ICD activity through posttranslational modification. Interestingly, Rv2170
is also capable of succinylating and propionylating ICD (using succinyl-CoA and propionyl-CoA
donors), suggesting that these modifications might also play a physiological role (45). Reversible
acetylation of Acs and propionyl-CoA synthetase (Ms5404) has also been shown to regulate both
acetate and propionate metabolism in M. smegmatis (29).
THE GLYOXYLATE SHUNT AS A DRUG TARGET

Recent work has shown that flux through the glyoxylate shunt also plays an important role in
pathogenicity. For example, mutants of M. tuberculosis defective in ICL activity show impaired
survival in vivo (59). Similarly, Fahnoe et al. (21) reported that the glyoxylate shunt is conditionally
essential for the survival of P. aeruginosa in mammalian systems; an aceA glcB double mutant was
completely cleared from the lung tissue in a mouse pulmonary infection model just 48 h after
infection. This is significant because P. aeruginosa consistently occupies a slot in the Top Ten lists
of major public health threats worldwide, and new antipseudomonal drugs are urgently required.
ICL is also required for S. enterica persistence in a mouse chronic infection model, although
interestingly, the enzyme plays no apparent role in acute infections (22).
Nature has already targeted the glyoxylate shunt as an antimicrobial strategy; the human en-
zyme Irg1 synthesizes copious quantities of the ICL inhibitor itaconate during macrophage acti-
vation. However, P. aeruginosa (along with other pathogens such as Yersinia pestis) has also acquired
322 Dolan · Welch

the ability to degrade itaconate and can even live off this compound as a sole carbon source. Indeed,
this three-gene catabolic pathway is known to be crucial for the survival of Y. pestis in macrophages
(66). This is somewhat counterintuitive given that deletion of the ICL-encoding gene in Y. pestis
prevents growth on acetate but has no obvious effect on pathogenesis in a mouse model (67).
Given its importance in pathogenicity, and also that mammals do not encode orthologs of ICL
or MS, the glyoxylate shunt enzymes have become attractive targets for drug discovery. Several
inhibitors of ICL have been identified, including itaconate, bromopyruvate, nitropropionate, ox-
alate, and malate (30, 52). However, these are pharmacologically unsuitable for use in vivo because
they are toxic and nonspecific. For instance, nitropropionate also inhibits succinate dehydrogenase,
a pivotal enzyme of the TCA cycle (3). However, and in an effort to further exploit the glyoxylate
shunt enzymes for antimicrobial drug discovery, ICL and MS from a variety of pathogens have
been structurally elucidated (54, 68, 72), facilitating structure-based drug design approaches.
A textbook example of structure-based drug design is illustrated by the development of potent
phenyl-diketo acid inhibitors of MS from M. tuberculosis. A focused library of 35 glyoxylate-

like compounds was screened for their ability to inhibit MS. Of these, 19 (all phenyl-diketo
acids) exhibited inhibitory activity. Subsequent structure-activity relationship analyses revealed

that substitution at the ortho position of the phenyl ring increased stability and improved the IC50
and many of the compounds inhibited growth of the organism when applied at low-micromolar
concentrations (39). The most potent inhibitor [Z-methyl 4-(2-chloro-6-fluoro-3-methylphenyl)-
2-hydroxy-4-oxobut-2-enoate] exhibited high specificity in vivo and yielded a significant reduction
in bacterial load in a murine acute tuberculosis infection model (39).
Another promising compound, 2-vinyl-D-isocitrate (2-VIC), developed as a mechanism-based
inactivator of M. tuberculosis ICL, provides an alternative approach toward inhibiting flux through
the glyoxylate shunt. ICL-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate,
2-vinylglyoxylate, which forms a covalent adduct with the thiolate form of Cys191 in the active site.
Analogs of 2-VIC would be expected to exert little toxicity in mammalian cells, which presumably
lack the ability to generate 2-vinylglyoxylate (because they do not encode ICL) (64).
Perhaps a more remarkable observation was made by Fahnoe et al. (21), who identified sev-
eral new compounds capable of preventing the growth of P. aeruginosa on acetate as a sole car-
bon source. The same compounds had no effect on growth in glucose medium, suggesting that
(a) they are cell permeable and (b) they specifically target acetate metabolism. Moreover, the top
hit compounds prevented growth of a variety of pathogenic bacteria on acetate. Remarkably, when
tested in vitro for their ability to inhibit purified P. aeruginosa ICL and MS, the compounds were
found to inhibit both enzymes, indicating that a single drug can simultaneously target the two
consecutive enzymes in the glyoxylate shunt. However, the precise mechanism of inhibition and
specificity of these compounds has yet to be rigorously evaluated.
It was recently proposed that instead of ICL or MS, other peripheral enzymes could be more
appropriate targets for bringing about inhibition of the glyoxylate shunt. For instance, the kinase-
phosphatase AceK may be a good candidate, since its absence would promote carbon flux through
the TCA cycle (70). However, and as outlined elsewhere in this review, the E. coli model of branch
point flux does not apply to all pathogens, especially those that do not encode an AceK homolog
(e.g., M. tuberculosis).
FLUX THROUGH THE GLYOXYLATE SHUNT AFFECTS BACTERIAL

VIRULENCE FACTOR PRODUCTION, PERSISTENCE, AND
ANTIBIOTIC RESISTANCE
Beyond carbon assimilation, the glyoxylate shunt has been implicated in the response to a multitude
of stresses encountered during infection, such as hypoxia, starvation, and antibiotic challenge. For

example, disruption of the M. tuberculosis icl gene attenuated bacterial persistence and virulence
in immune-competent mice (53) and deletion of the ICL-1- and ICL-2-encoding genes together
led to a complete block on intracellular replication and rapid elimination from the lungs (58).
However, this may be only indirectly linked with the glyoxylate shunt, since ICL-1 also functions
as a 2-methylisocitrate lyase, a function that is critical for the catabolism of propionate (25).
Given that sources of propionate are abundant in biology, and that propionate and its metabolic
derivatives are toxic to cells, it is clear that disruption of the propionate catabolic pathway could
impair the fitness of the organism. Therefore, the consequences of inactivating ICL are potentially
complex and extend beyond its canonical role in the glyoxylate shunt (20, 76).
Using a clever genetic system in which M. tuberculosis MS could be temporally depleted through
doxycycline-dependent transcriptional silencing and anhydrotetracycline-dependent proteolytic
degradation of MS, Puckett et al. (65) showed that the number of tuberculous lung lesions in
a mouse infection model declined dramatically in the absence of MS. Interestingly, they also
noted that a mutant in glcB (the gene encoding MS in M. tuberculosis) grew well on glucose in
vitro, but displayed a pronounced growth defect in the presence of oleic acid, an effect that could
be overcome by addition of the ICL inhibitor itaconate. This indicated that MS is required to
resist fatty acid–associated toxicity and that this toxicity likely arises from accumulation of the
glyoxylate generated by ICL (65). Subsequent metabolic profiling revealed that the glcB mutant
also accumulated ketone bodies, indicative of a backlog of acetyl-CoA and depletion of cellular
oxaloacetate levels. However, the exact mechanism of glyoxylate toxicity remains to be elucidated.
Metabolomic profiling was carried out on M. tuberculosis to discover a common set of metabolic
changes associated with the activities of three clinically relevant tuberculosis drugs, isoniazid,
rifampicin, and streptomycin. All three drugs led to a remodeling of central metabolism and,
in particular, yielded a 4- to 16-fold increase in icl transcription. Consistent with the likely role
played by ICL in this, an icl mutant was over 100-fold more susceptible to all three antibiotics; a
phenotype that could be rescued by chemical complementation of the cultures with an antioxidant.
It seems, then, that in M. tuberculosis ICL participates in an endogenous metabolic defense against
the bactericidal consequences of antibiotic-induced oxidative stress, although the mechanistic
details remain to be elucidated (61). However, this is not the case in all organisms. Deletion of the
ICL-encoding gene (aceA) in P. aeruginosa improved cell growth under conditions of oxidative and
antibiotic stress (1). Glyoxylate shunt–deficient P. aeruginosa mutants also underwent a metabolic
shift toward aerobic denitrification, indicating that ICL may also play a role in regulation of
respiration and ROS management (1). Flux through the glyoxylate shunt can also impinge on
antibiotic resistance in other ways. Aminoglycoside uptake is dependent upon a proton-motive
force (PMF), set up as a consequence of electron transport chain activity. Supplementation of
P. aeruginosa cultures with glyoxylate confers protection against aminoglycoside (tobramycin)
toxicity, apparently by allowing the cell to bypass the NADH-producing steps of the TCA cycle,
thereby lowering the PMF and reducing uptake of the drug (55). Drugs that target the glyoxylate
shunt may therefore also act as adjuvants, increasing the therapeutic activity of existing antibiotics.
Flux through the glyoxylate shunt also impinges on virulence factor production. Many bacterial
pathogens use the type III secretion system (T3SS) to inject toxic effector proteins directly into
eukaryotic cells, thereby subverting host function (28). The expression and activity of the T3SS are
normally stimulated by direct contact with host cells. However, under some circumstances, the host
cell contact requirement is bypassed and the T3SS becomes controlled by metabolic inputs. For
example, during microaerobic growth of P. aeruginosa, expression of the T3SS becomes strongly
stimulated, even in the absence of host cells. Crucially, this stimulation of the T3SS is dependent
upon the activity of ICL. Interestingly, the T3SS in P. aeruginosa is normally under the control
of a signaling pathway governed by the activity of a kinase, RetS, at the apex. ICL activity is low
324 Dolan · Welch

in a retS mutant, and complementation of the retS mutant with aceA in trans not only restores
ICL activity but also restores the T3SS (12). Consistent with the probable role played by ICL in
infection scenarios, clinical isolates of P. aeruginosa frequently overexpress ICL (47), and RNAseq
analyses have shown that aceA is 25-fold upregulated during infection in a mouse model (75).
The role of the glyoxylate shunt in the photosynthetic cyanobacteria is less clear. Cyanobacterial
metabolism is adapted for phototrophic growth, with the photosynthetic electron transport chain
as the main source of reducing agents and ATP in the presence of light. One possibility is therefore
that the glyoxylate shunt is required for dark reactions. This seems unlikely per se because acetate
does not support sustained heterotrophic growth of Cyanothece strain 7424 in the absence of
light. However, ICL activity is induced by acetate when Cyanothece 7424 is grown under diurnal
day/night conditions, and cultures grown with acetate exhibit increased biomass production (26).
Clearly, more work is needed in this important area.
METABOLON FORMATION AND SUBSTRATE CHANNELING

A metabolon is a temporary complex of (often sequential) enzymes in a pathway. Such complexes

ensure rapid transfer of substrates between active sites, bypassing the necessity for diffusion-limited
intersite transfer and thereby maintaining maximal flux with a minimal number of enzymes and
with concomitant protection of labile intermediates (5, 9, 85). Considering the reactivity and
toxicity of glyoxylate, it is understandable why such a system would be favored. Unfortunately,
there is a paucity of published work addressing this issue experimentally in microbial systems,
especially given that one would predict robust metabolon formation between ICL and MS in
order to avoid intracellular accumulation of glyoxylate. This has indeed been observed in the
glyoxysomes of Zea mays, but not in bacteria (6).
METABOLIC ENGINEERING—APPLICATIONS OF THE

GLYOXYLATE SHUNT
Under normal circumstances, the glyoxylate shunt is irreversible—it only operates in the acetyl-
CoA assimilating direction and is incapable of producing acetyl-CoA from dicarboxylates. This
irreversibility is due to the MS-catalyzed reaction; the ICL reaction is readily reversible in vivo.
Because of this irreversibility, the most common sugars can only be metabolized to acetyl-CoA via
decarboxylation of the three-carbon molecule pyruvate, representing a major loss of carbon from
the system as CO2 . A pathway to convert malate and succinate to oxaloacetate and two molecules
of acetyl-CoA was recently engineered in E. coli. This was accomplished by incorporating malate
thiokinase (Mtk) (which converts malate to malyl-CoA in an-ATP catalyzed reaction) and malyl-
CoA ligase (Mcl) (which converts malyl-CoA to acetyl-CoA and glyoxylate). If further integrated
with central metabolism, this pathway has the potential to form the basis of several novel carbon
fixation cycles (50, 77).
However, our favorite manipulation of the glyoxylate shunt—if only because of its sheer bold-
ness and curiosity-driven ethos—has to be the study of Dean et al. (19), who investigated what
happens when the glyoxylate shunt is introduced into mammals. Unlike bacteria and the select
few eukaryotic genera that encode a functional glyoxylate shunt, mammals generally lack any way
of converting acetyl-CoA to sugar. Sadly then, and unlike most bacteria, we humans cannot live
off vinegar alone. It is therefore a fascinating intellectual problem to ask what happens if we allow
mammals to carry out such a transformation—and this is exactly what Dean et al. (19) did. Con-
trary to expectation, the expression of E. coli aceA (ICL) and aceB (MS) in mouse liver led to an
increase in fatty acid degradation and a decrease in total fat mass accumulation. Moreover, mice

expressing the shunt showed resistance to diet-induced obesity even on a high-fat diet, despite
having similar food consumption as the controls (19). Thus, the full potential of the glyoxylate
shunt in metabolic engineering will be realized in the coming years.
PARTING COMMENTS—LOOKING TO THE FUTURE

As outlined in this review, the regulation of carbon flux partitioning between the glyoxylate shunt
and TCA cycle has been investigated in detail for only two bacterial species, E. coli and M.
tuberculosis, and both of these clearly use very different mechanisms to control fluxes. There is
no one-size-fits-all solution, and in spite of its historical significance and major contribution to-
ward our understanding of many fundamental aspects of biochemistry, the E. coli mechanism of flux
control is less of a paradigm than it once was. Indeed, with the genomic revolution in full swing,
opportunities abound for novel regulatory mechanisms to be identified, as well as novel actors
(witness the emergence of acetylation as a regulatory posttranslational modification over the last
decade). Perhaps future work should focus on characterizing these noncanonical systems where
AceK either is not present (Table 1) or cannot be the principal regulatory player. For example, like
M. tuberculosis, P. aeruginosa encodes both ICD and IDH isozymes, yet unlike M. tuberculosis, this
organism also encodes a functional AceK. Clearly, neither phosphorylation alone nor allostery
is likely to account for flux control in such a complex system. Given that the glyoxylate shunt
underpins pathogenicity in some of the world’s most threatening bacterial pathogens, and given
that the shunt can potentially be targeted for therapeutic intervention, a better understanding of
its biochemistry and enzymology will be important.
SUMMARY POINTS
The metabolic bifurcation between the glyoxylate shunt and the TCA cycle is one of the
most important branch points in the whole of microbiology.
Flux partitioning between the glyoxylate shunt and the TCA cycle is governed by the
enzymology at the branch point.
Genome sequencing and biochemical analyses indicate that the enzymology governing
isocitrate dissimilation is likely to vary widely between organisms and cannot necessarily
be extrapolated from a detailed understanding of the well-characterized Escherichia coli
model.
Flux partitioning can be regulated by posttranslational modification of the enzymes in-
volved (phosphorylation, acetylation, etc.), and through allostery involving low molecular
weight metabolites.
The glyoxylate shunt is an essential pathway in many pathogens and an excellent target
for the development of antimicrobial compounds.
Virulence factor production and antibiotic susceptibility are strongly influenced by gly-
oxylate shunt activity.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
326 Dolan · Welch

ACKNOWLEDGMENTS
Work on the glyoxylate shunt and propionate catabolism in the M.W. laboratory is supported
by the BBSRC, the European Union, and the Jardine Foundation. S.K.D. is a BBSRC-funded
postdoctoral fellow.
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The Glyoxylate Shunt, 60 Years On
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Communication Between the Microbiota and Mammalian Immunity
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Regulation of Sexual Commitment and Gametocytogenesis
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Pneumococcal Vaccines: Host Interactions, Population Dynamics,
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Indexes
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Errata
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MI72CH15_Welch ARI 11 August 2018 7:53

Annual Review of Microbiology

The Glyoxylate Shunt,

Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW,

Annu. Rev. Microbiol. 2018. 72:309–30 Keywords

TRANSCRIPTIONAL REGULATION OF THE GLYOXYLATE SHUNT IN

DIFFERENT BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

BACKGROUND AND A BRIEF HISTORY OF THE GLYOXYLATE SHUNT

310 Dolan · Welch

Fumarate CH CH2 COO–

www.annualreviews.org • The Glyoxylate Shunt 311

THE TEXTBOOK MECHANISM OF TCA CYCLE–GLYOXYLATE

In E. coli, NADP-dependent isocitrate dehydrogenase (ICD) catalyzes the conversion of iso-

CONTROL OF AceK KINASE/PHOSPHATASE ACTIVITY IN E. COLI

312 Dolan · Welch

STRUCTURAL INSIGHTS INTO THE MECHANISM OF E. COLI AceK

www.annualreviews.org • The Glyoxylate Shunt 313

THE aceBAK OPERON: GENETIC CONTEXT AND TRANSCRIPTIONAL

MICROBIAL PATCHWORK—INSIGHTS FROM 20 YEARS

314 Dolan · Welch

MS ICL AceK IclR

ATP ADP Inactive

www.annualreviews.org • The Glyoxylate Shunt 315

IS THE E. COLI REGULATORY MODEL REPRESENTATIVE OF OTHER

316 Dolan · Welch

Staphylococcus aureus ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ citC ∗∗∗ NA

Helicobacter pylori HP0027 NA

www.annualreviews.org • The Glyoxylate Shunt

Klebsiella pneumoniae KPNIH24_01265 KPNIH24_01270 KPNIH24_18460 KPNIH24_01260 aceBAK operonic

TRANSCRIPTIONAL REGULATION OF THE GLYOXYLATE SHUNT IN

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a RccR regulon in Pseudomonas spp. b RamB/PrpR regulon in Mycobacterium spp.

RccR RccR Propionate

POSTTRANSLATIONAL REGULATION OF THE GLYOXYLATE SHUNT

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replaced by an amber (TAG) stop codon. The N -acetyllysyl-tRNA synthetase/tRNACUA pair of

THE GLYOXYLATE SHUNT AS A DRUG TARGET

322 Dolan · Welch

phenyl-diketo acid inhibitors of MS from M. tuberculosis. A focused library of 35 glyoxylate-

acids) exhibited inhibitory activity. Subsequent structure-activity relationship analyses revealed

FLUX THROUGH THE GLYOXYLATE SHUNT AFFECTS BACTERIAL

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324 Dolan · Welch

METABOLON FORMATION AND SUBSTRATE CHANNELING

A metabolon is a temporary complex of (often sequential) enzymes in a pathway. Such complexes

METABOLIC ENGINEERING—APPLICATIONS OF THE

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PARTING COMMENTS—LOOKING TO THE FUTURE

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328 Dolan · Welch

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bacteria. Eur. J. Biochem. 176(3):497–508

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Volume 72, 2018 Contents

The Outer Membrane Took Center Stage

M. Daniel-Ivad, S. Pimentel-Elardo, and J.R. Nodwell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p25

Above and Beyond Watson and Crick: Guanine Quadruplex Structures

The Promise of a Malaria Vaccine—Are We Closer?