J. Mol. Microbiol. Biotechnol. (2002) 4(3): 269–276. JMMB Symposium

The Molecular Biology of Formate in Enterobacteria

Susanne Leonhartsberger, Ingrid Korsa, Formate Metabolism and August Bo¨ ck* The key reaction in the switch of aerobic carbohydrate Institute of Genetics and Microbiology, University of degradation to is the cleavage of pyr- Munich, Maria-Ward-Strae 1a, D-80638 Munich uvate (Figure 1). Aerobically and also under nitrate respiratory conditions, pyruvate is oxidised by pyru- Abstract vate dehydrogenase (Kaiser and Sawers, 1994). Upon shift to anaerobic growth in the absence of external Formate is the signature compound in the anaerobic electron acceptors pyruvate is cleaved predominantly metabolism of and other entero- by pyruvate-formate- (PFL) in a non-oxidative . Its synthesis and degradation is integrated reaction thus yielding acetyl-CoA and formate. Both in a net-work of metabolic routes which are ele- the activity and the synthesis of PFL are subject to gantly regulated to adjust the carbon flux to the complex regulation. In aerobically grown cells PFL is metabolic needs. This review summarises the present in a basal amount but in an inactive form. Shift information on the biochemistry of synthesis and to anaerobiosis leads to activation of the by degradation of formate, on the genetics of the converting it into a radical form via abstraction of members of the regulon and on the mechanism ahydrogen atom from glycyl residue 734. Flavodoxin underlying the regulation. is functioning as an electron donor and S-adenosyl- methionine as (for review see Kessler and Knappe, 1997). Formate is a key metabolite in the energy metabolism PFL H S adenosylmethionine flavodoxin of many bacteria. Because of its low redox potential À þ À þ red ! of 420 mV it can be oxidised not only through the PFL 50 desoxyadenosine À þ À aerobic respiratory chain but it can also serve as an methionine flavodoxinox electron donor for the anaerobic reduction of fumarate þ þ (Macy et al.,1976) and of nitrate or nitrite (Wimpenny The three-dimensional structure of PFL was determined and Cole, 1967). The prominent role of formate in energy recently in complex with the substrate analogon metabolism is particularily pronounced in the mixed- oxamate; it suggested the involvement of two cysteine acid-fermentation of enterobacteria since one third of residues in the reaction mechanism whereby one, the sugar carbon degraded is converted into formate. Cys418, serves as intermediary thiyl radical and Depending on the physiological condition, formate may Cys419 as H-atom donor (Becker et al., 1999; Plaga be secreted, oxidised aerobically, used as a reductant in et al., 2000). PFL in its glycine radical form is with a variety of terminal reduc- sensitive; when it is exposed to molecular oxygen the carbon backbone of the polypeptide is cleaved at the tases, or converted into CO2 and H2 via the formate hydrogen-lyase reaction (Stephenson and Stickland, site of the glycine radical. Activation of PFL is catalysed 1932; Ordal and Halvorson, 1939). In the latter physio- by a specific activase whose gene is located down- logical situation acidification is counteracted by the stream of the pfl structural gene. When fermentatively conversion of the into a neutral molecule, i.e. growing cells of E. coli are shifted to aerobic conditions, hydrogen, which possesses a redox potential compar- theradical form of PFL is deactivated in a hitherto able to that of formic acid. All these reactions are neatly uncharacterised reaction catalysed by AdhE, the tied together in a regulatory network, designated for- enzyme reducing acetyl-CoA to ethanol with acetalde- mate regulon, in which formate itself plays a decisive hyde as an intermediate (Kessler and Knappe, 1997). role as effector molecule (Rossmann et al., 1991). In this Concomitant with activation of the enzyme during review we shall deal first with an outline of the ashift from aerobiosis to fermentative growth condi- biochemical reactions involved in formate production tions an approximately 12 to 15-fold induction of PFL and consumption and with the transcriptional units synthesis takes place. Besides elongation factor Tu whose expression is under the control of formate. PFListhus the most abundant protein in fermenting Finally, we shall concentrate on the regulation of cells. Factors involved in the induction process are formate metabolism and discuss the knowledge avail- theFnr protein, the ArcAB two-component regulatory able on the main regulators involved, the FhlA and HycA system and the integration host factor (Sawers and proteins. Watson, 1998; Alexeeva et al., 2000). As a conse- quence of the high-level induction and the activation, actively fermenting E. coli cells accumulate formate

*For correspondence. Email [email protected]; levels up to 20 mM in their cytoplasm (cited in Sawers, Tel. +49(0)89-2180-6120; Fax. +49(0)89-2180-6122. 1994).

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Order from caister.com/order 270 Leonhartsberger et al.

Figure 1. Synthesis and degradation of formate in the metabolism of E. coli.Thepathways are shown schematically. THF: tetrahydrofolate, Ac-CoA: acetyl-CoA, Prop-CoA: propionyl-CoA, Met-Prot.: proteins with N-terminal methionines, GAR: glycinamide ribonucleotide, F-GAR: formyl glycinamide ribonucleotide. For details see text.

Recently, a second anaerobic ketoacid formate- nities for formate, namely 0.12 mM for FDH-N and lyase has been discovered in E. coli.Itisinvolvedin 26 mM for FDH-H (Enoch and Lester, 1975; Axley anaerobic threonine metabolism and cleaves 2-keto et al., 1990; for detailed review see Unden and butyrate into propionyl-CoA and formate (Hesslinger Bongaerts, 1997). Intracellularly generated formate et al., 1998). Moderate contributions to the formate can be readily exchanged with the environment. Two levels in E. coli are also provided by the posttransla- gene products were identified with a function in tional deformylation of N-formyl-methionine residues formate transport. They are FocA which is the pro- and during purine biosynthesis (Figure 1). duct of the first gene in the pfl transcriptional unit In addition, formate serves as the hydrogen donor (Suppmann and Sawers, 1994) and FocB, a product of for the ribonucleoside-triphosphate reductase respon- one of the genes of the hyf operon from E. coli sible for the production of deoxyribonucleoside triphos- (Andrews et al., 1997). Experimental evidence for such phates during anaerobic growth of E. coli (Mulliez et al., atransport function has been provided for FocA but not 1995). yet for FocB. Formate, therefore, is present both in aerobically and anaerobically grown cells; accordingly, both aero- Genetics of Formate Metabolism bic and anaerobic degradation routes have evolved (for detailed review see Sawers, 1994 and Unden and E. coli harbours four transcriptional units on its Bongaerts, 1997). Aerobic cells express a formate chromosome whose expression is determined by the (FDH-O) first described by Pinsent intracellular concentration of formate (Figure 2A). (1954); it appears to couple formate oxidation with fdhF:The monocistronic fdhF gene codes for the ubiquinone reduction in the respiratory chain. The selenopolypeptide of FDH-H. It is transcribed from a enzyme is also present under nitrate respiratory condi- sigma-54 dependent promoter whose activation re- tion (Sawers et al., 1991). The predominant enzyme quires the presence of an upstream regulatory se- in nitrate reducing cells, however, is FDH-N. The quence (UAS) centered between 100 to 140 membrane-bound enzyme has been purified by Enoch nucleotides upstream of the transcriptionÀ startÀ site and Lester (1975) and well characterised in different (Birkmann and Bo¨ ck, 1989; Birkmann et al., 1987). groups (see Sawers, 1994). Its synthesis is induced hyc genes: The hycBCDEFGH genes code for during anaerobiosis and by nitrate. FDH-H finally, is a components of the formate hydrogen-lyase systems component of the formate hydrogen-lyase complex of which accept electrons from FDH-H and reduce protons E. coli.Itisloosely associated with the inner side of the to molecular hydrogen (see Figure 2B) (Bo¨ hm et al., cytoplasmic membrane and is readily detached during 1990; Sauter et al., 1992). HycB displays sequence breakage of the cells. FDH-H was purified by Axley similarity with the small subunit of formate dehydro- et al. (1991) and its three-dimensional structure has genases and could serve as the membrane docking been resolved (Boyington et al., 1997). All three target of FDH-H, HycC and HycD are membrane- formate dehydrogenases are selenoproteins carrying integral proteins with a putative anchoring function amolybdopterin dinucleotide cofactor. Of relevance for and HycE and HycG are the large and small subunits of the discussion of the regulatory aspect is that FDH-N hydrogenase 3, respectively. Judged from sequence and FDH-H possess greatly different apparent affi- signatures, HycF appears to be an electron carrier Formate Regulon 271

Figure 2. Organisation of the genes and structural model of the FHL complex in E. coli.A.Thefourtranscriptional units of the formate regulon. Big arrows represent genes, small boxes UAS sequences, small arrows promoters. Genes coding for structural components of the FHL complex are shown in black, genes of accessory proteins in grey and regulatory genes in white. Hyd: hydrogenase. Functions of the gene products are shown where biochemical evidence was presented. B. Model of the FHL complex associated to the membrane and the functions of its components. Subunits of the hydrogenase 3 are shaded in dark grey. but its position within the redox chain is unknown. The promoter-proximal genes of this operon code for function of HycH which is required for generation of proteins involved in the maturation of hydrogenase formate hydrogen-lyase activity is unknown. As dis- 3(hypA, hypC)orofall three hydrogenases (hypB, cussed later, HycA is involved in regulation and HycI in hypD, hypE)(forreviewsee Maier and Bo¨ ck, 1996). the maturation of the complex. The hyc operon is The ultimate gene of this operon is fhlA which codes expressed from a sigma-54 dependent promoter (Pc) forthe central regulator of the formate regulon (Sankar located upstream of hycA cooperating with an UAS et al., 1988; Schlensog and Bo¨ck, 1990). The regula- positioned in the center of the hyc-hyp intergenic region tion of the expression of this operon implies the activity (Lutz et al., 1990). of three promoters. PP is located 50 to hypA and its The hyp operon is inversely oriented to the activation is sigma-54 dependent. Interestingly, its hyc transcriptional unit; the product of the five corresponding UAS is positioned in the intergenic 272 Leonhartsberger et al.

Table 1. Physiological conditions influencing the expression of the genes of the formate regulon and the experimental evidence for the involvement.

Factor Experimental Evidence Reference

Formate no expression in Dpfl strain, formate induces Birkmann et al., 1987 2 2 Molybdate no expression in mod strain, MoO4 À and WO4 À induce Schlensog et al., 1989 Rosentel et al., 1995 Oxygen or nitrate external electron acceptors repress expression of fdhF::lacZ or hyc::lacZ Pecher et al., 1983 pH < 7expressionoffdhF::lacZ or hyc::lacZ correlates with gas formation only at acidic pH Rossmann et al., 1991

region between hycA and hycB,some800 bp more promoters. As a consequence, FDH-H and the other upstream (Hopper et al., 1994). A second promoter PA components of the formate hydrogen-lyase system are is located within the hypA gene; it acts with sigma-70 synthesized and their activity counterbalances a further and requires the Fnr protein for activation (Lutz et al., increase of formate by converting it into CO2 and H2 1991). Finally, there is a weak and constitutively active (Rossmann et al., 1991). promoter in the intergenic region between hypE and How does the pH signal tie in? Initially it was fhlA. argued that the effect of acidification relies on the Afourth transcriptional unit whose expression is redistribution of formate from the extracellular space responsive to formate is located downstream of the hyc into the cytoplasm, possibly as a proton driven import operon and consists of two genes, hydN with a putative (Rossmann et al., 1991). However, in view of the pKa role in electron transfer and hypF with a function in value of formate of about 3.4 this possibility appears the maturation of all three hydrogenases (Maier et al., not plausible since full induction of the expression is 1996). reached already at a pH value of 6.0 The resulting increase in the protonated form of formate should be insufficient to increase the intracellular concentrations Physiological Conditions Affecting Expression to avalue above 1 mM. of the Formate Regulon Molybdate dependence of the expression of the genes coding for hydrogenase 3 seems to be mediated The expression of the genes of the formate regulon by different systems. The ModE protein has been shown depends on a considerable number of physiological to be a required component in the activation of the hyc conditions (Peck and Gest, 1957; Wimpenny and Cole, operon to an optimal level in the presence of formate 1967; Pecher et al., 1983). They are summarised in (Self et al.,1999). Additionally, MoeA is involved in the Table 1, together with the experimental evidence. activation of the hyc operon (Hasona et al., 1998) and it Expression is absolutely dependent on the presence has been suggested that it catalyses the production of of formate and molybdate and is counteracted by the an activated form of molybdate, which in turn might terminal electron acceptors oxygen and nitrate. Fuma- interact with the transcriptional activator FhlA (Self and rate exerts only a moderate effect. Finally and most Shanmugam, 2000). importantly, the genes of the regulon are only induced at apHinthemediumlower than 7.0. Rossmann et al. (1991) presented a model in which Mechanism of Action of FhlA the effects of formate, oxygen, nitrate and pH are all integrated to represent the effect of a single effector FhlA belongs to the NtrC group of transcriptional molecule, namely formate (Figure 3). Under aerobic regulators which consist of an N-terminal domain A, a conditions, PFL is inactive and formate is not present at conserved central domain C and a C-terminal domain D. significant concentrations since FDH-O efficiently con- Domain C – which contains an ATP – is sumes the low amounts formed via the non-fermenta- thought to interact with RNA polymerase x sigma-54 tive routes (see Figure 3). Upon shifting E. coli cells to and domain D has been shown to interact with the UAS fermentative growth conditions, PFL is activated and with its helix-turn-helix motif. The three domains are induced in its synthesis and formate is produced. At usually separated by short linkers (Drummond et al., neutral pH the majority of it is excreted, possibly under 1986). involvement of the activity of the FocA protein. At more FhlA belongs to a subclass of these regulators acidic pH, the scenario depends on the relative affinities which share with the NtrC prototypes only the structural of formate for FDH-N and FDH-H and for the central features of the C and D domains (Schlensog and Bo¨ ck, regulator of the formate regulon FhlA. When nitrate is 1990). This subclass which also includes NifA, DmpR present, FDH-N is induced and effectively oxidises and XylR lack an aspartyl residue at the A domain formate because of its superior Km.Intheabsence of which is the site of phosphorylation in the classical nitrate, formate accumulates until it reaches a concen- NtrC-type regulators. Regulators of this type do not tration sufficient to bind to FhlA, which has an apparent appear to require phosphorylation by a sensor kinase binding constant of 5 mM (Hopper and Bo¨ ck, 1995). but liganding of a small molecule to be converted into FhlA in complex with the ligand formate then binds to the the activated form. In the case of XylR and DmpR these UAS and activates transcription from the sigma-54 effector molecules are aromatic compounds (Shingler Formate Regulon 273

Figure 3. Different metabolic systems competing for the degradation of formate under different conditions. For details see text.

and Moore, 1994), in the case of FhlA it is formate. tion that the FhlA variants which activate transcription Experimental proof that formate per se activates has in the absence of formate show the same kinetics of been provided by in vitro transcription-translation ATP hydrolysis as wild-typeFhlAinthe presence of the assays and by analysing the ATPase activity of purified effector (Hopper and Bo¨ ck, 1995; Korsa and Bo¨ ck, FhlA in response to formate and its UAS (Hopper et al., 1997). When the complete domain A was removed, the 1994; Hopper and Bo¨ ck, 1995). Moreover, azide, a truncated FhlA showed high and constitutive ATP structural analogon of formate, can mimick its effect. hydrolysis as well as transcriptional activation whereas Mutants of FhlA have been selected that activate the domain A in trans reduced the activity of full-length transcription in the absence of formate and it has been FhlA, indicating (i) that domain A – like that in other shown that the exchanges are located in regulators – modulates the binding and hydrolysis of domain A of FhlA (Korsa and Bo¨ ck, 1997). Although a ATP, and (ii) that domain A binds formate (Leonharts- structure of FhlA with its effector is not yet available, all berger et al., 2000). It also supports the notion that these results show that formate binds to FhlA most FhlA does not need phosphorylation to be active. probably in the N-terminal domain A. However, as the activity of the N-terminally truncated The predominant functional consequence of the FhlA is still stimulated in the presence of UAS, binding of formate is a significant stimulation of the activation by DNA and formate seem to be at least ATP hydrolysis activity due to a decrease of the KM of partially independent processes. FhlA for ATP. Interestingly, an increase of the ATP Purified FhlA interacts with both the specific DNA concentration resulted in ligand – independent ATP (carrying the UAS) and unspecific DNA (lacking the hydrolysis, which supports the conjecture that the UAS) as judged by stimulation of ATPase activity. modulation of the ATPase activity is the key task of the However, when both formate and DNA ligands are ligand. This assumption is supported by the observa- offered, only the specific DNA triggers a drastic 274 Leonhartsberger et al.

Figure 4. Regulation of the expression of the genes of the formate regulon under aerobic (A) and anaerobic (B) conditions. For details see text. stimulation of the kinetics of ATP hydrolysis (Hopper analyses revealed features of FhlA that allowed the and Bo¨ ck, 1995). It is at present unknown whether the proposition of a model in which FhlA undergoes a increase in ATPase activity is a direct consequence of change in its oligomeric state for transcription activa- the binding of formate and/or DNA or whether the tion and that this oligomerisation is favored by high ligands change the oligomerisation state of the protein nucleoside triphosphate concentrations, by the effec- which leads to the observed changes in ATPase torformate and by the target DNA (Hopper et al., activity. Such an oligomerisation has been shown 1996). to be crucial for the activity of other regulators of the Recently, the DNA-binding site of FhlA was NtrC family (Weiss et al., 1992). In vitro transcription analysed in detail. Each UAS consists of two binding Formate Regulon 275 sites of 16 bp length with the consensus sequence conditions there is an autoinduction of the expression 50-CATTTCGTACGAAATG-30 separated by a spacer of fhlA since transcription initiates at PP and continues region. Hydroxyradical footprinting analysis identified over fhlA.PromoterPA is active under anaerobic six sites on each strand within the UAS which are bound conditions in the absence of high formate concentra- by FhlA (Leonhartsberger et al., 2000). Formate and tions; its physiological relevance appears to reside in ATP had no detectable influence on the binding pattern. the provision of the hyp gene products for the Amodel was proposed in which FhlA binds the DNA on maturation of hydrogenases 1 and 2 (Lutz et al., 1990). only one side of the helix, probably as a tetramer. In The autoinduction of fhlA expression is counter- vivo experiments with constructs in which insertions acted by the formation of HycA as an antiactivator and shifted the binding sites on different sites of the helix by the formation of formate hydrogen-lyase which confirmed this model (Leonhartsberger et al., 2000). decreases the formate concentration. An intriguing additional issue is that fhlA transla- The Role of HycA as Antiactivator tion is under the control of OxyS (Altuvia et al., 1998). The production of this 109 nucleotide untranslated When a mutation was introduced into hycA –the RNA is induced in response to oxidative stress in promoter-proximal gene of the hyc operon – the E. coli.It has been shown that OxyS RNA inhibits fhlA formate hydrogen-lyase activity was increased two- translation by pairing with a sequence overlapping the fold. When hycA was overexpressed in trans,the ribosome binding site and a short sequence within the activity was decreased to undetectable levels (Sauter coding region of fhlA.Kissingcomplex formation et al., 1992). Clearly, this behavior indicates that HycA between OxyS and fhlA results in a stable antisense- has a regulatory role counteracting the activity of FhlA. target complex (Argaman and Altuvia, 2000). Oxidative As hycA is part of the hyc operon the amount of the stress, like a shift from fermentation to aerobic protein in the cell in turn is regulated by FhlA and conditions, as characteristic for the life style of E. coli, formate. Theoretically, there are two levels at which thus can immediately shutdown synthesis of the HycA might act. First, HycA might interact with FhlA via regulator and block wasteful formation of the oxygen- protein-protein contact and down-modulate its activity; sensitive formate hydrogen-lyase system. secondly, HycA could be involved in the control of the cellular formate concentration, e.g., by functioning as Acknowledgement an export facilitator which assists in extruding formate. The work in the authors laboratory summarized in this review has been At present, the issue is unresolved. It is also unknown continuously supported by grantsfrom the Deutsche Forschungsge- whether HycA directly senses some extra- or intracel- meinschaft and the Fonds der Chemischen Industrie. lular signal. References

Related Systems Alexeeva, S., de Kort, B., Sawers, G., Hellingwerf, K.J., and de Mattos, M.J. 2000. Effects of limited aeration and of the ArcAB Klebsiella species ferment glucose essentially along the system on intermediary pyruvate in Escherichia coli. same routes as E. coli but they produce predominantly J. Bacteriol. 17: 4934–4940. Altuvia, S., Zhang, A., Argaman, L., Tiwari, A., and Storz, G. 1998. neutral end products like ethanol and butanediol. The The Escherichia coli OxyS regulatory RNA represses fhlA translation three involved in the conversion of two moles by blocking ribosome binding. EMBO J. 17: 6069–6075. of pyruvate into one mole of butanediol are organised in Andrews, S.C., Berks, B.C., McClay, J., Ambler, A., Quail, M.A., one operon (budABC)(forreviewsee Magee and Golby, P., and Guest, J.R. 1997. A 12-cistron Escherichia coli operon (hyf)encoding a putative proton-translocating formate Kosaric, 1987). In Klebsiella terrigena expression of hydrogenlyase system. Microbiology 143: 3633–3647. the bud operon is regulated by BudR which is encoded Argaman, L., and Altuvia, S. 2000. fhlA repression by OxyS RNA: by a gene located in inverse orientation to budABC and Kissing complex formation at two sites results inastableantisense- separated from it by a 106 bp intergenic region (Mayer target RNA complex. J. Mol. Biol. 300: 1101–1112. Axley, M.J., Bo¨ ck, A., and Stadtman, T.C. 1991. Catalytic properties of et al., 1995). BudR is a LysR-type regulator and an Escherichia coli formate dehydrogenase mutant in which sulfur specifically responds to the acetate concentration in replaces selenium. Proc. Natl. Acad. Sci. USA 88: 8450–8454. the medium. Acidic pH and anaerobiosis stimulate Axley, M.J., Grahame, D.A., and Stadtman, T.C. 1990. Escherichia coli formate-hydrogen lyase: purification and properties of the the effect of acetate. It appears that BudR coordinates selenium-dependent formate dehydrogenase component. J. Biol. the activity of the energy-conserving, non-reductive but Chem. 265: 18213–18218. acidifying acetate formation pathway with that of the Becker, A., Fritz-Wolf, K., Kabsch, W., Knappe, J., Schultz, S., and non-energy-conserving and non-acidifying butanediol Wagner, A.F.V. 1999. Structure andmechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 10: 969–975. pathway (Mayer et al., 1995). Birkmann, A., and Bo¨ ck, A. 1989. Characterisation of a cis regula- tory DNA element necessary for formate induction of the formate Synposis dehydrogenase gene (fdhF)ofEscherichia coli.Mol.Microbiol.3: 187–195. Birkmann, A., Zinoni, F., Sawers, G., and Bo¨ ck, A. 1987. Factors Figure 4 summarises the scenarioes of the expression affecting transcriptional regulation of the formate-hydrogen-lyase of the genes of the formate regulon under aerobic (part pathway of Escherichia coli.Arch. Microbiol. 148: 44–51. A) and fermentative (B) conditions. Aerobically, fhlA is Bo¨ hm, R., Sauter, M., and Bo¨ ck, A. 1990. Nucleotide sequence and solely transcribed from its constitutive promoter; due to expression of an operon in Escherichia coli coding for formate hydrogenlyase components. Mol. Microbiol. 4: 231–243. the lack of PFL activity the genes of the formate Boyington, J.C., Gladyshev, V.N., Khangulov, S.V., Stadtman, T.C., regulon are not induced. Under fermentative growth and Sun, PD. 1997. Crystal structure of formate dehydrogenase H: 276 Leonhartsberger et al.

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