Anaerobic Dissimilation of Pyruvate
Chapter
15
DOROTHEA KESSLER and JOACHIM KNAPPE
A considerable number of fermentation end products are observed when Escherichia coli cells grow anaerobically with glucose as carbon and energy source. The list includes succinate, lactate, acetate, ethanol, formate, carbon dioxide, and dihydrogen. Although the route to succinate is initiated at the stage of phosphoenolpyruvate, all the other compounds are derived from pyruvate (Fig. 1). Product patterns vary considerably, depending on the specific conditions. With dioxygen strictly excluded and the pH maintained at about pH 7, batch-cultured E. coli K-12 (in glucose minimal medium) does not form dihydrogen and produces only traces of lactate (3). Under these conditions, the products formed (in millimoles per 100 mmol of glucose) are about as follows: succinate, 12; acetate, 75; ethanol, 87; and formate, 113; 3 g (dry weight) of cell mass, corresponding to 115 meq of carbon, is also formed.
The emphasis in this chapter is on the enzymology of the steps of pyruvate conversion to acetate and formate as follows (CoA is coenzyme A):
pyruvate + CoA
acetyl-CoA + formate (1)
acetyl-CoA + Pi
acetyl phosphate + CoA (2)
acetyl phosphate + ADP
acetate + ATP (3)
This reaction sequence represents the backbone of the cellular machinery for the anaerobic life of E. coli. The enzyme catalyzing the reaction in equation 1, pyruvate formate-lyase (PFL), is tightly controlled on the transcriptional as well as on the posttranslational level. Its high catalytic activity in the anaerobic cell makes this enzyme a key element in governing the glucose fermentation route.
The succession of the three reactions to be discussed, with individual Δ G o' values of –3.9, +2.2, and –3.1 kcal/mol (1 cal = 4.184 J), respectively (data are from reference 55), also represents the most lucid example of substrate level phosphorylation, since there is no redox chemistry involved. In fact, the E. coli cell is sustained solely by the single ATP gained in this manner when the cell is grown anaerobically with pyruvate as sole carbon and energy source (39). In bioenergetic terms, pyruvate is classified as a high-energy compound. The (acetyl) group transfer potential Δ G o' (of pyruvate hydrolysis to acetate and formate; calculated from the values given above and –7.6 kcal for ATP hydrolysis) is –12.4 kcal/mol. Pyruvate is thus energetically equivalent to its usual metabolic precursor, phosphoenolpyruvate.
The properties and cellular levels of the proteins involved in pyruvate conversion to acetate are summarized in Table 1.
Table 1Proteins involved in pyruvate dissimiliation (E. coli K-12) |
PFL (EC 2.3.1.54), a homodimer from a constituent polypeptide of 759 amino acids, is identified as a radical enzyme (28, 61). The active form harbors the glycine 734 as an α-carbon centered radical, displaying a characteristic electron paramagnetic resonance spectrum (g = 2.0037) and UV absorption (365 nm). Secondary-structure predictions suggest its location in a β-turn segment (-Val-Ser-Gly-Tyr-), where the unpaired electron can delocalize optimally over the adjacent coplanar oriented peptide units, which affords the thermodynamic stabilization of this π-type radical (17) (Fig. 2). Though kinetically stable under anaerobic conditions, the glycyl radical moiety is extremely sensitive to oxygen, which makes tight control of PFL status in the E. coli cell (i.e., over the active form and its nonradical precursor form) mandatory. Oxygen contact produces specific scission of the polypeptide backbone at site 734, thereby converting the glycyl into an oxalyl group. It was this modification that originally indicated the location of the protein-based radical in the primary structure (61).
PFL catalyzes the reaction in equation 1 very efficiently; the turnover numbers (at 30C) for the forward and reverse directions are 770 and 260/s, respectively (25). The overall catalytic cycle comprises two discrete half-reactions that involve an isolatable acetyl enzyme intermediate (also carrying the glycyl radical), where Cys-419 acts as the covalent catalytic SH (25, 40):
Acetyl phosphinate (
) as a pyruvate analog and hypophosphite (
) as a formate analog are mechanism-based irreversible inhibitors of PFL (28, 61). They react specifically with E•-SH and E•-S-acetyl, respectively, yielding 1-hydroxyethyl phosphonate that is linked to Cys-418 [CH3-CHOH-PO(O–)-S-Cy] (40). A carbon-centered radical of this common dead-end product has been characterized as a reaction intermediate (57).
The results from these suicide reactions and the specific defectiveness of Cys-418 mutants (C418S or C418A) in cleaving the pyruvate substrate are strong evidence that the SH group of Cys-418, which is adjacent to the central Cys-419, also participates directly in the crucial first half-reaction of the catalytic cycle (27). An intermediary thiyl radical of Cys-418 which promotes substrate processing by initially forming an adduct to the carboxyl group of pyruvate (or formate), thus governing carbon-carbon bond cleavage and annealing along a radical chemical route, is suggested. (This mechanism is described in detail in reference 27.)
The operational organization of the radical enzyme PFL is envisaged now as comprising Gly-734 as the site where the unpaired electron is located in the enzyme’s resting state, whereas the utilization of the unpaired electron in catalysis occurs in the active-site region of the central Cys-Cys pair. These separate sites would be linked by (long-range) electron transfer reactions, which are triggered by substrate binding (forward direction) and product release (reverse direction):
The radical-chemical mechanism of PFL for carbon-carbon bond cleavage or synthesis is unique among enzymatic reactions of cellular metabolism, which are dominated by ionic processes.
The dissimilation of pyruvate into acetate (acetyl-CoA) and formate by E. coli cell extracts was discovered in the laboratories of C. H. Werkman and F. Lipmann (8, 22, 58) as early as 1943 or 1944; this scientific era is associated also with the disclosure of the functional significance of the novel coenzyme A, highlighted by the structural identification of its acetyl thioester in 1951 by F. Lynen (34). The unusual (bio)chemistry of the pyruvate cleavage reaction did not emerge in a direct way by studying active PFL enzyme isolated straightforwardly from the anaerobic cell extract. Instead, further work in the 1960s (outlined in reference 29) usually employed aerobic cells for enzyme fractionation and used anoxic conditions only for assay purposes. This approach yielded a set of four proteins together with adenosylmethionine (AdoMet) (identified quite early as a crucial cofactor [26, 31]), which was then found to operate as an electron donor system (flavodoxin) and a converter enzyme reaction that transformed an inactive 170-kDa protein into the actual catalyst of the PFL reaction (25, 30). The elusive nature of the protein modification, not recognized immediately as an amino acid radical by protein chemical analyses, was finally revealed through the electron paramagnetic resonance signal displayed by active PFL (28, 61).
Notably, flavodoxin as electron carrier and the novel biochemistry of AdoMet discovered with PFL conversion are now recognized as being involved in further enzyme activation processes in E. coli (see The Flavodoxin-Ferredoxin System in E. coli, below).
Production of the PFL radical (Fig. 3) according to the equation
E-H + AdoMet + e– → E• + 5'-deoxyadenosine + methionine
is mediated by a monomeric protein, PFL activase, that requires Fe2+ as cofactor (28, 29, 30); the metal-binding site could involve a cluster of Cys residues with spacings identical to that in the Fe protein Fnr. The activase reaction is (virtually totally) dependent on the presence of pyruvate or the oxamate analog (25), which has a conformational effect on the nonradical-form PFL substrate (A. F. V. Wagner and J. Knappe, unpublished data). Glycyl radical synthesis occurs by stereoselective abstraction of the Gly-734 proS hydrogen atom, which becomes incorporated into the 5'-deoxyadenosine coproduct (17). A 5'-deoxyadenosine-5'-yl radical is proposed as the immediate H atom abstractor, generated in the active site of PFL activase from AdoMet and dihydroflavodoxin (which last is replaceable in vitro by artificial one-electron reductants). Recognition of the Gly-734 residue in PFL apparently occurs solely through a short amino acid sequence, since activase transforms even heptapeptides that are homologous to this site (Arg-Val-Ser-Gly-Tyr-Ala-Val) (17).
A distinct converter enzyme, PFL deactivase, which is capable of transforming active PFL back to the nonradical form, is also contained in E. coli (29). Molecular cloning disclosed that it is identical to the AdhE protein (23, 24), which normally performs the terminal NAD regeneration in glycolysis, i.e., reduction of acetyl-CoA to ethanol (Fig. 1). This multienzymic protein, requiring Fe2+ for all functions, is a polymer of 96-kDa subunits with a helical assemblage into rods 60 to 200 nm long (Fig. 4); it should be identical to the "spirosomes" formerly detected in various facultative and obligate anaerobes by electron microscopists (35). A specific requirement of CoA and NAD (with conversion to NADH) is established for the glycyl radical reduction by AdhE (23), but the mechanism is unresolved.
PFL exists virtually totally in the radical form in the E. coli cell grown anaerobically on glucose (11), although AdhE protein is present in abundance. Suppression of the deactivation function of AdhE protein probably occurs through the high cellular levels of pyruvate and NADH; these metabolites have been identified as inhibitors in the in vitro reaction (23). PFL deactivation, however, takes place when anaerobic cell cultures are shifted to redox potentials of the medium of ≥100 mV (23). Conversely, PFL protein that is expressed during aerobic cell growth (about 10% of the anaerobic level) is held totally in the nonradical form (11, 29), which should be primarily because of the lack of dihydroflavodoxin reductant. It is evident that intracellular control devices in E. coli that are yet to be delineated cope perfectly with the protein-destructive oxygen sensitivity of the active PFL enzyme.
Both types of the widely distributed small (and highly acidic) proteins that contain either flavin mononucleotide (flavodoxin) or 2Fe-2S (ferredoxin) for promoting one-electron transfer from a highly negative potential occur constitutively in E. coli (60). Flavodoxin was originally discovered through its function in PFL activation; ferredoxin was detected at that time (through its red-brown color) as a by-product in large-scale procedures for isolating flavodoxin. Two reductases that afford electron transfer to each of these proteins have been identified (6). The thiamin diphosphate-dependent pyruvate:flavodoxin/ferredoxin oxidoreductase (CoA acetylating) has not been examined in the homogenous state or genetically mapped but functionally resembles well-characterized enzymes from other bacteria (a compilation is given in reference 5). The other reductase, encoded by the fpr gene, is a flavoprotein using NADPH or (less efficiently) NADH as electron donor substrate.
Cobalamin-dependent methionine synthase of E. coli (MetH) requires activation in which (dihydro)flavodoxin reduces cob(II)alamin and AdoMet methylates the cob(I)alamin, generating the catalytically competent methylcobalamin enzyme form (2). (The flavodoxin system components involved here were formerly described [18] as R and F.) Anaerobic ribonucleotide reductase (NrdD), recently characterized as a further glycyl-free radical enzyme, is transformed to the active state by a flavodoxin-dependent and AdoMet to 5'-deoxyadenosine-converting process (41) which closely resembles the PFL system. Conversion of dethiobiotin to biotin, mediated by biotin synthase (BioB), also requires flavodoxin plus AdoMet (14, 21), which, considering these other systems and chemical feasibility, could suggest involvement of radicals.
Contrary to the various electron donor roles (in posttranslational enzyme modifications) known for flavodoxin, no such function has been documented so far for the ferredoxin species. The spectral characteristics and amino acid sequence of this 2Fe-2S cluster protein (32, 54) show striking similarities to the ferredoxin from Pseudomonas putida (putidaredoxin) and the mammalian mitochondrial adrenodoxin that participate in cytochrome P-450 reactions, which, however, are unknown for E. coli.
The reversible acetyltransfer reaction catalyzed by phosphotransacetylase (EC 2.3.1.8) (equation 2) occurs, according to results with the clostridial enzyme (20), through direct displacement mechanisms between the substrates. Arsenate can replace phosphate, and any acetyl arsenate formed is spontaneously hydrolyzed. This property is utilized in a classic assay of CoA and phosphotransacetylase (CoA-dependent "arsenolysis" of acetyl phosphate) (51).
The dimeric E. coli enzyme (48) is cold labile, dissociating into inactive protomers. The activity is effected positively by pyruvate and negatively by NADH (53) through the increase and decrease, respectively, of the enzyme affinity for acetyl-CoA according to a K-type allosteric mechanism (H. P. Blaschkowski and J. Knappe, unpublished data).
Acetate kinase (EC 2.7.2.1), which reversibly catalyzes the reaction in equation 3 (43), is also a cold-labile dimeric protein (16). It appears to be unorthodox among phosphotransferases (and transferases in general) with respect to the catalytic mechanism. The steric inversion determined for the γ-phosphoryl group of ATP at transit to acetate (7) indicates a direct phosphoryl transfer (involving ternary complexes of substrates and enzyme). On the other hand, acetate kinase is well documented (1, 16) as forming a phosphoenzyme (with a phosphorylated glutamyl [56]) readily and reversibly from ATP or acetyl phosphate. Whether this represents an obligatory reaction intermediate (inferring even a triple-displacement mechanism of the catalytic route [50]) is a matter of controversy.
In any case, the "autophosphorylating" acetate kinase has recently gained significance in other functional directions. The phosphoenzyme can replace phosphoenolpyruvate for phosphorylating enzyme I of the phosphotransferase sugar transport system (15), and acetyl phosphate and acetate kinase/phosphotransacetylase are suggested as representing important components of an internal metabolic regulation network in E. coli (12, 37, 62).
The key enzyme of pyruvate dissimilation, PFL, is expressed during anaerobic growth at a more than 10-fold-increased level (11), which occurs by induction of pfl gene transcription with unique features (Fig. 5). Sawers and Bck (46) identified seven tandemly oriented transcription start sites for σ 70 RNA polymerase which are commonly activated in strictly coordinate fashion by an upstream regulatory region that is recognized by the two global transcription regulators Fnr and ArcA as well as by the integration host factor. The Fnr protein affords the major fraction of pfl gene activation by anaerobic growth conditions (47); integration host factor mediates the further induction observed with pyruvate additions to the growth medium (49). (How the regulator proteins can effect downstream promoters 1 through 5, from which transcription in fact starts most frequently [46], is an intriguing question yet to be resolved [45].)
The pfl gene forms an operon with a second gene, focA, whose product is a 31-kDa membrane protein that exports formate from the cell (52).
Significantly increased protein synthesis during anaerobic growth of E. coli also occurs with the AdhE protein (10, 24). The upstream region of the adhE gene contains a potential Fnr recognition sequence (24), suggesting transcriptional control as for the pfl gene; this matter is unresolved (adhE gene expression patterns are analyzed in reference 9). Mutants that lack AdhE require an exogenous electron acceptor (nitrite or nitrate) for anaerobic growth in glucose minimal medium (39), which demonstrates the fundamental NADH reoxidation function of this multienzyme in the glycolysis route.
PFL activase, which is encoded by the act gene located close to the pfl gene (42, 44), and flavodoxin and its reductases are synthesized constitutively. Their copy numbers in wild-type K-12 cells (each about 3,000) are about that of the PFL protein that is expressed even in aerobic growth (29). Hence, active PFL enzyme can be made very quickly once the cells are exposed to an anaerobic environment.
The high activity of PFL in strictly anaerobically growing E. coli K-12 wild-type cells is the reason that the possible glycolysis route to lactate is normally (at neutral or alkaline pH) bypassed completely. Pure lactate fermentation, through the terminal d-lactate dehydrogenase (LdhA) reaction, occurs in mutant cells that are defective in PFL; they require the addition of acetate to grow anaerobically (39).
E. coli is equipped with the complete set of three enzyme systems that convert pyruvate to acetyl CoA and occur in nature. The oxidative enzymes, the pyruvate dehydrogenase complex and pyruvate:ferredoxin/flavodoxin oxidoreductase, commonly involve thiamin diphosphate as coenzyme for carbon-carbon bond cleavage, yielding CO2 as coproduct. PFL with the glycyl-free radical content functions by a fundamentally different mechanism and has clearly evolved independently. The fact that the PFL reaction is readily reversible leads to the speculation that the original function was perhaps in the opposite, anabolic direction. Its roots could go back to a prebiotic thioester world (13) with a greater abundance of formic acid, as suggested by the Miller spark discharge experiments.
The energetically profitable (and chemically most elegant) pyruvate dismutation pathway is exploitable by E. coli, since this facultative anaerobe has acquired an apparently perfect system of transcriptional and postribosomal control to allow for the oxygen sensitivity of the glycyl radical element. The external-internal signal network involved herein remains to be delineated.
The work on PFL conducted in this laboratory was supported by grants from the Deutsche Forschungsgemeinschaft.
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Chapter15
