Glycerol and Methylglyoxal Metabolism

IAN R. BOOTH

[SECTION EDITOR: AUGUST BÖCK]

Posted March 29, 2005

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom

Phone: 44-6224-555852, Fax: 44-6224-555824, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Glycerol and methylglyoxal (MG) are peripherally related. They share a metabolic branchpoint since dihydroxyacetone phosphate (DHAP), which is an intermediate in the aerobic metabolism of glycerol, is the precursor of MG. However, while glycerol can be considered to be an important carbon source, the synthesis of MG is a consequence of unbalanced metabolism related either to a limitation for phosphate or to excessive carbon flux through the pathways that have the capacity to generate significant pools of DHAP. Cells producing MG must either detoxify or die. It is here that the intrinsic fascination of MG biosynthesis lies: Cells produce a poison as an intermediate strategy for survival of metabolic imbalance. Indeed the panoply of metabolic regulation in this sector of catabolism can be seen as a strategy to avoid death by self-poisoning.

INTRODUCTION

Glycerol metabolism represents a relatively simple cluster of biochemical activities leading to 3-phosphoglyceraldehyde, the entry point to the lower section of glycolysis prior to metabolism by the tricarboxylic acid (TCA) cycle (Fig. 1). However, glycerol-3-phosphate (Gly-3-P), the first intermediate in the pathway, is also the point of convergence with both the biosynthesis and catabolism of lipids. Phosphodiesterases in both the periplasmic (glpQ) and cytoplasmic (ugpQ) compartments liberate Gly-3-P from phospholipids, enabling the cell to recycle this important intermediate in both energy metabolism and biosynthesis. External Gly-3-P is the inducer of the glp genes and its accumulation in the cytoplasm is carefully controlled to avoid toxicity. Despite the simplicity of the pathways there is significant regulation both of the synthesis of the enzymes and the activity of glycerokinase. In part, this may reflect the potential of glycerol metabolism to perturb cell viability through provision of a direct route to the synthesis of methylglyoxal (MG). The latter compound is synthesized from dihydroxyacetone phosphate (DHAP), which is the product of oxidation of Gly-3-P. Most bacterial species have evolved multiple pathways for detoxifying MG; Escherichia coli and Salmonella enterica serovar Typhimurium are no exception to this rule. However, one detoxification pathway, the glutathione-dependent glyoxalase system, dominates. In addition, the activity of this pathway is linked directly to the activation of protective K⁺ efflux systems, KefB and KefC. Recent work has identified these pathways of protection and detoxification as being at the heart of the response of serovar Typhimurium cells to entry into the phagosome (12).

Fig. 1Glycerol metabolism and its relationship to methylglyoxal synthesis. The major enzymatic routes in aerobic cells are indicated by solid arrows. A broken arrow is used to indicate the anaerobic Gly-3-P oxidation pathway. The methylglyoxal synthesis pathway is boxed to indicate the special nature of this aspect of the metabolism. Standard nomenclature is used to indicate the genes for the enzymatic steps.

GLYCEROL TRANSPORT AND METABOLISM

Glycerol entry into E. coli and serovar Typhimurium is facilitated by the aquaglyceroporin, GlpF (23, 44). A homologous protein in serovar Typhimurium, PduF, facilitates the entry of 1,2-propanediol (Ppd) and is part of the Ppd metabolic pathway (2). GlpF expression is coregulated with the enzymes of glycerol catabolism. In the absence of GlpF the rate of glycerol entry into cells is slow and such mutants exhibit a glycerol-negative growth phenotype when grown at low glycerol concentrations (43). Growth is normal at higher concentrations of the triol because of the intrinsically high passive permeability of the lipid bilayer. The glpF gene is part of the glpFKX operon that also encodes glycerol kinase (GlpK) and GlpX, an alternative fructose-1,6-bisphosphatase that has a potential role in gluconeogenesis. Although the GlpF protein acts as a pore to facilitate entry to equilibrium, it has been proposed that GlpK is physically associated with GlpF, leading to rapid phosphorylation of glycerol as it enters the cell. GlpK is subject to noncompetitive inhibition by fructose-1,6-bisphosphate and by enzyme III^Glc of the PEP-dependent phosphotransferase system (7, 8, 9, 10, 30, 38). This control leads to a very effective block on glycerol metabolism in the presence of glucose as a result of the higher concentration of the two inhibitors. It is the release from these inhibitory influences that potentiates MG production during growth on glycerol.

The alternative fructose-1,6-bisphosphatase, GlpX, is inhibited by fructose-1-phosphate, ADP, and phosphate and activated by phosphoenolpyruvate; this implies a potential role in gluconeogenic growth (11). However, no physiological role has been discerned for GlpX at the level of expression normally observed in cells, since there is an alternative enzyme (Fbp), which is required for gluconeogenic growth on glycerol (19). The glpX gene can act as a multicopy suppresser of an E. coli fbp mutant and chromosomal fbp suppresser mutations map to the glpFKX operon (11). These data are consistent with coregulation of glpX expression with the other enzymes of glycerol metabolism encoded by the glpFKX operon, precluding a more general role in gluconeogenesis.

Externally generated (or supplied) Gly-3-P is transported by the GlpT transporter that exchanges Gly-3-P for phosphate and, in this way, the cell avoids the toxicity associated with accumulation of either Gly-3-P or inorganic phosphate. The crystal structure of the GlpT transporter has recently been described and a molecular mechanism for the exchange proposed (25). In the cytoplasm the transported Gly-3-P is oxidized by one of two membrane-bound flavin-dependent oxidases encoded by glpD and glpABC for the aerobic and anaerobic complexes, respectively. In the presence of oxygen or nitrate the GlpD enzyme transfers electrons to respective terminal oxidases. Under anaerobic conditions the GlpABC system transfers the electrons to fumarate. In the absence of fumarate, expression of glpABC is limited even in the presence of Gly-3-P; addition of fumarate to the growth medium leads to increased expression of the dehydrogenase complex (48). The inability of E. coli to grow on glycerol or Gly-3-P under anaerobic conditions is due to its inability to oxidize the carbon source in the absence of fumarate as an electron acceptor. Consequently, cells exposed to glycerol under anaerobic conditions accumulate high levels of Gly-3-P to a point where it becomes inhibitory to growth, and this condition is relieved by fumarate (48).

Glycerol can also be oxidized directly by glycerol dehydrogenase (gldA; Gdh) generating dihydroxyacetone (DHA) with the concurrent reduction of NAD⁺ (39). In Klebsiella pneumoniae glycerol kinase can also phosphorylate DHA, permitting growth on DHA in the absence of DHA kinase, but this does not appear to be a significant pathway in E. coli (26). Recently homologues of DHA kinase have been detected in the genome of E. coli leading to the proposition of the potential for DHA phosphorylation at the expense of phosphoenolpyruvate (PEP) (27). In K. pneumoniae the dha operon ensures the fermentation of glycerol, leading to production of 1,3-propanediol (Ppd) (18, 45). Parallel oxidative and reductive metabolic pathways ensure the conversion of two molecules of glycerol to one each of DHAP, which enters glycolysis, and one of Ppd, which is excreted from the cell. The oxidative branch utilizes NAD⁺-dependent Gdh and PEP-dependent DHA kinase. A B₁₂-dependent glycerol dehydratase (GDHt) converts glycerol to 3-hydroxypropionaldehyde (3-HPA), which becomes the substrate for NADH-dependent 1,3-propanediol oxidoreductase (PDOR) (Fig. 2) (18). NADH that is produced by the oxidative branch of glycerol breakdown is reoxidized by the reductive branch of the pathway (Fig. 2). Glycerol dehydratases are unusual in undergoing irreversible inactivation by the substrate glycerol, and also by oxygen, followed by reactivation by a specific chaperone complex DdrAB (44, 46). Inactivation involves the cleavage of the cobalamin-adenosyl bond in B₁₂, causing release of 5'-deoxyadenosine and tight binding of the alkylcobalamin. The DdrAB reactivation complex catalyzes the exchange of the enzyme-bound deadenylated cobalamin for free adenine-containing cobalamins in a two-step process requiring both ADP and ATP (29, 36).

Fig. 2Anaerobic glycerol metabolism in Klebsiella. Broken arrows are used to indicate the fates of the dihydroxyacetone phosphate and 1,3-propanediol. Gdh, glycerol dehydrogenase; GDHt, glycerol dehydratase; PDOR, 1,3-propanediol oxidoreductase; DHA kinase, PEP-dependent dihydroxyacetone kinase; PEP, phosphoenolpyruvate.

This pathway provides a further link to MG production in recombinant E. coli (49). Introduction of the cloned dha genes into E. coli simultaneously allowed the production of 1,3-propanediol and anaerobic growth on glycerol. High concentrations of glycerol inhibited growth due to the intracellular accumulation of Gly-3-P and MG production. Coexpression of Pseudomonas putida glyoxalase I in the recombinant E. coli strains increased the capacity of detoxification of MG and simultaneously increased the anaerobic growth on glycerol and Ppd production (49).

METHYLGLYOXAL SYNTHESIS AND DETOXIFICATION

The metabolic connection between glycerol and MG is principally that DHAP, which is an intermediate in the aerobic breakdown of glycerol, is also the major precursor of MG, being the substrate for methylglyoxal synthase (MGS) (24). MG is a potent electrophile. Its accumulation at submillimolar concentrations leads to growth inhibition and cell death due to modification of proteins and DNA (31). In E. coli K-12 MG production from glycerol requires mutations that alter the regulation of expression of glpFKX operon genes coupled with release of glycerol kinase from feedback inhibition (20). Normally, the pathway is subject to inducer exclusion and allosteric control acting on GlpK to regulate flux into the pathway. Additionally, MG synthesis is prevented by allosteric inhibition of MGS by phosphate and by the maintenance of low intracellular pools of DHAP, since the synthase is homotropically activated by DHAP (6). An increase in the DHAP pool coupled with depletion of the cytoplasmic phosphate pool predisposes cells to produce MG. These conditions are known to arise in the laboratory in mutants in which the regulation of carbon-utilizing pathways is modified. Although MG production can occur as a side reaction of triose phosphate isomerase (TPI) and synthesis from amino acids and other metabolites (6), the dominant enzyme in MG production is MGS (47).

MGS catalyzes the elimination of phosphate from DHAP, forming an enzyme-bound enediol(ate) intermediate that is released from the enzyme, followed by release of inorganic phosphate. The enzyme is highly specific for DHAP (6, 41). The mgsA gene (22 min on the E. coli chromosome) (47) produces a 17-kDa monomer, but the crystal structure, resolved to 1.9 Å, has shown the enzyme to be a homohexamer (40). Analysis of the homotropic activation by DHAP suggested a Hill coefficient of ~3 for DHAP, which led to the original proposal of a tetramer (24), but which is also consistent with the hexamer (40). The allosteric regulation of MGS is extremely tight since E. coli cells expressing ~10³-fold greater enzyme activity due to the induced expression of the cloned mgsA gene displayed only low levels of MG production (47). This represents the balance of synthesis and detoxification in a cell with 1,000 times the normal synthetic activity and indicates that the rate of MG production is extremely low during "normal" growth on glucose.

Rapid synthesis of MG is potentiated by inactivation of feedback control over enzyme activity and subversion of regulatory mechanisms controlling gene expression (1, 20, 28, 47). Loss of catabolite repression over enzyme synthesis for pathways that feed into glycolysis at DHAP or 3-phosphoglyceraldehyde stimulates MG production in the presence of the substrates of these pathways. E. coli cya mutants, which lack adenyl cyclase activity, frequently acquire hypersensitivity to cAMP when secondary mutations arise that suppress the original defect in catabolite repression. Such mutants produce abundant MG upon exposure to cAMP, leading to cell death (32). Similarly, cultures growing on d-xylose, l-arabinose, or d-glucose 6-phosphate are stimulated to produce MG when exposed to cAMP to suppress self-(catabolite) repression (1, 47). Substrate uptake is frequently the rate-limiting step under self-repression, and addition of cAMP increases transport system expression such that the cell is flooded with substrate. An imbalance between the induced pathway and relatively fixed activity of glycolysis leads to lowering of the cytoplasmic phosphate pool and accumulation of precursors of DHAP because of the consequent metabolic bottleneck at GA3P dehydrogenase. Catabolite repression serves to maintain an appropriate balance between the inducible and semiconstitutive pathways (1, 28, 47). Two modifications to the glycerol pathway are required to precipitate MG synthesis: constitutive expression of the glp operon and a glycerol kinase that is insensitive to feedback inhibition by fructose 1,6,-bisphosphate (20). In addition, prior growth on carbon sources that generate only weak catabolite repression, such as succinate or casein amino acids, which maximizes the expression of the glp operon (20), is an important precondition for MG production.

MGS is not an essential enzyme under most laboratory conditions (47). Mutants of E. coli lacking MGS exhibit essentially normal growth patterns until challenged with unbalanced metabolism (47). Synthesis of MG from DHAP and its detoxification by the glyoxalases to produce d-lactate led to the pathway being deemed the MG bypass of glycolysis (6). Enzyme kinetics support this proposal, since MGS should be inhibited as long as the P_i concentration is similar to the K_m of GA3P dehydrogenase (24). Phosphate limitation would lead to inhibition of GA3P dehydrogenase, would cause increased DHAP pools via TPI activity, and would simultaneously relieve the inhibition on MGS leading to MG production. The synthesis of MG provides a mechanism of liberating phosphate and allowing further carbon flow through glycolysis with the attendant production of carbon intermediates and ATP. When subjected to excess carbon source influx, cells possessing MGS continue to grow and therefore have the potential for further adaptation. In contrast, an mgsA mutant of E. coli undergoes immediate and severe growth inhibition (47). Analysis of gene expression after entry of serovar Typhimurium into macrophages identified increased expression of the genes encoding phosphate-scavenging systems, glyoxalase I and KefB (12). Limitation of E. coli cells for phosphate leads to MG production at a rate that outstrips the capacity for detoxification (47). In E. coli, glyoxalase I and KefB are limiting for detoxification and protection (31), which makes sense of the expression patterns seen in serovar Typhimurium, and it may be that the need for the MGS and glyoxalase pathways is greatest under conditions where pathogens are invading host tissues.

Multiple MG detoxification pathways are found in both E. coli and serovar Typhimurium, but the dominant pathway is the GSH-dependent glyoxalase I-II system (31) (Fig. 3). Two other systems for MG detoxification are the GSH-independent glyoxalase III (34) and an oxidoreductase (21, 35) (Fig. 3). Neither makes a major contribution to detoxification under normal circumstances. In E. coli, where mutants lacking GSH or glyoxalase I (GlxI) have been studied, the dominant pathway has been shown to be the GSH-dependent glyoxalase I-II pathway, encoded by the gloA and gloB genes (31). Mutants lacking either GSH or GlxI exhibit only slow MG detoxification and are extremely MG sensitive. Overexpression (~30-fold) of GlxI activity leads to an approximately 3-fold increase in MG breakdown, despite an increase of up to 100-fold in the specific activity of the enzyme. This difference almost certainly reflects limitations on the pathway imposed by the need to recycle GSH via GlxII activity and the reaction between MG and GSH in the cytoplasm. However, this increase in GlxI activity is sufficient to exert considerable additional protection (31). Overexpression of GlxII does not accelerate the rate of MG detoxification but, because of its inhibitory effect on KefB activity (see below), leads to impaired viability in the presence of MG (31).

Fig. 3Methylglyoxal detoxification. See text for explanation. A strong dotted line is used to indicate the activation of KefB by S-lactoylglutathione. Fine dotted lines indicate the glutathione-independent methylglyoxal detoxification pathways. These two pathways (GlxIII and MGR) remain poorly characterized at the genetic level but appear to be quantitatively insignificant. Standard genetic acronyms are used for the genes encoding the major enzymes of the pathway. GSH, glutathione.

Cytoplasmic GSH reacts with MG to generate hemithiolacetal, which is the substrate for GlxI. The product of the isomerization, S-lactoylglutathione, is the substrate for GlxII, which regenerates GSH and releases the product d-lactate. The gloA and gloB genes are unlinked on the E. coli chromosome, lying at 36 min and 5 to 6 min, respectively (31). The structure of GlxI protein from E. coli has been solved and demonstrated to be a Ni²⁺-dependent enzyme (4, 22). Genes that appear to encode GlxI proteins have been identified in many bacteria through analysis of the genome projects (5, 42). Glyoxalase II has been much less intensively studied but the structural gene was cloned and expressed in E. coli (31). It is possible that there are two distantly related homologues of this protein in both E. coli and serovar Typhimurium. The second homologue in E. coli YcbL (~20 min on the E. coli genetic map) has not been characterized in detail. Mutants lacking GlxII have not been isolated. There have been no systematic studies of their regulation on any of the enteric bacteria, but gene array experiments have identified increased expression of gloA in Salmonella after entry into macrophages, implying some level of control (12). S-Lactoylglutathione is an activator of the KefB K⁺ efflux system and leads to protection of the cell until detoxification is complete (31).

The KefB and KefC systems have evolved to provide protection during detoxification of electrophiles (13, 14, 15, 16, 17, 31). KefB and KefC are GSH-gated K⁺ efflux systems that are activated by the formation and binding of glutathione adducts that are generated during detoxification. The proteins are highly similar in their sequence, organization, and regulation (3). Mutations introduced at the equivalent positions in the respective genes of each protein have essentially similar effects (33, 37). KefB provides the greatest protection against MG and is activated by the product of GlxI, S-lactoylglutathione (16, 31). As a result of activation of KefB (or KefC) E. coli cells release K⁺ and the ions are replaced by Na⁺ and H⁺. The resulting acidification of the cytoplasm is sufficient to protect against electrophiles. Activation of KefB and KefC causes an approximately 0.4 to 0.6 pH unit acidification of the cytoplasmic pH. The final cytoplasmic pH achieved in the first 20 to 30 min after exposure to MG is critical to survival. Activation of KefB and KefC causes an initial undershoot of the cytoplasmic pH on the acid side, the magnitude of which correlates with the level of channel activity (31). This is followed by a small rise in the cytoplasmic pH. Good protection is achieved if the cytoplasmic pH lies below pH 7.5 (14, 15, 16). Channel activation can be mimicked by lowering the cytoplasmic pH with weak organic acids and even GSH-deficient mutants, which are very sensitive to MG due to the lack of the GlxI-II pathway, are protected by the lowering of the cytoplasmic pH with organic acids. Mutants lacking KefB and KefC are unaltered in their rate of detoxification despite the loss of cell viability, and the rate of detoxification is unaltered even after the culture has lost greater than 95% viability (15).

KefB activity is influenced by GlxI and GlxII activity (31). Initial studies suggested that KefB exhibited much less activity than KefC but was more specific for the GSH adducts formed during MG detoxification (16, 31). Mutants lacking KefB and KefC are sensitive to MG, but the effect is quantitatively less than the effect of the loss of GlxI. Expression of higher levels of GlxI was found to stimulate KefB activity and mutants lacking GlxI were not activated by incubation with MG. These data suggested that KefB is activated by the product of GlxI activity, S-lactoylglutathione (SLG). This model was supported by the observation that overexpression of GlxII diminished KefB activation and inhibited survival during exposure to MG (16). This is predicted from the model (Fig. 3), since extra GlxII will lower the cytoplasmic pool of SLG. Since the KefB system is maintained closed by binding GSH and is activated by SLG, the activity of the system is a reflection of the rate of synthesis of MG and its rate of detoxification. The observed induction of GlxI and KefB in serovar Typhimurium upon entry into macrophages makes sense in this model. Increasing GlxI should raise the rate of detoxification and increase the activity of KefB, thus improving cell survival. Clearly the systems integrate to provide an effective protection mechanism during the period of unbalanced metabolism caused by phosphate limitation.

Acknowledgments

This work was supported by The Wellcome Trust (Grant 040174).

I thank my colleagues who have contributed to this work during the past ten years. Special thanks are extended to Michelle Edwards for her careful reading of the manuscript.