The tricarboxylic acid cycle of Helicobacter pylori

Eur. J. Biochem. 260, 258±267 (1999) q FEBS 1999

The tricarboxylic acid cycle of Helicobacter pylori

Stuart M. Pitson1, George L. Mendz1, Sujatha Srinivasan1 and Stuart L. Hazell2

1School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia; 2School of Microbiology and Immunology,

University of New South Wales, Sydney, Australia

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter

pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry.

NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible

using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete

tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase,

aconitase, isocitrate dehydrogenase, a-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase,

and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle,

including a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown.

The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic

metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and a-

ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of

a-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational

glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a g-aminobutyrate shunt, owing to the

absence of both g-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic

and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino

acid sequences with those of other, more extensively studied enzymes.

Keywords: TCA cycle, Helicobacter pylori, NMR, microaerophily.

Helicobacter pylori is a gram-negative, microaerophilic bac-terium that commonly colonizes the upper gastrointestinal tractof humans, occurring in an estimated 50% of the world'spopulation [1]. It is the principal cause of chronic gastritis [2,3],duodenal and gastric ulcers [4], and has been linked with thedevelopment of gastric cancer [5±7]. Owing to its importance,H. pylori has been the focus of considerable research since itsfirst isolation in 1982 [8].

The complete H. pylori genome has been sequenced [9] andaspects of the pathogen's metabolism and physiology have beenelucidated [10], but the mode of operation of its bioenergeticand biosynthetic pathways remain mostly unknown. Unlike otherCampylobacter-like bacteria, H. pylori has the capacity to take upand metabolize glucose via the pentose phosphate and Entner-Doudoroff pathways [11±14]. The intermediary pyruvate andfumarate metabolism of the bacterium have been examined[15,16] revealing the presence of pyruvate-flavodoxin oxido-reductase [17] and fumarate reductase [18], respectively, as

potential targets for therapeutic intervention. The existence of denovo pyrimidine and salvage purine nucleotide synthesis pathways[19,20], the urea cycle [21], and other aspects of nitrogenmetabolism also have been studied [10]. Urease, catalase [22] andseveral other enzymes [15,18,23±25] have been characterized.Some elements of the tricarboxylic acid (TCA) cycle have beenfound through biochemical [18,26,27] and genomic analyses [9],although considerable uncertainty exists over the exact composi-tion in H. pylori of this central pathway; a problem this study hasaddressed.

The TCA cycle performs a dual role in cell metabolism,providing biosynthetic starting compounds such as a-ketoglu-tarate, succinyl-CoA and oxaloacetate, which act as precursorsfor a wide spectrum of cell components, as well as acting as a`metabolic energy' source through the generation of reducednucleotides, the reoxidation of which may be coupled to ATPsynthesis. Because of this dual role and the widespread occur-rence of the TCA cycle in nature, it is not surprising thatdifferent organisms, according to their needs, have placeddifferent emphasis on the metabolic functions of the cycle givingrise to a considerable diversity in its way of operating [28].H. pylori has the ability to take up and metabolize glucose [11±14], but it appears that it can utilize preformed amino acids andfatty acids present in the gastric mucosa [23], the natural habitatof the bacterium, to satisfy most of its metabolic energyrequirements. The carbon skeletons resulting from the catabol-ism of these molecules could enter the TCA cycle and bedirected to satisfy biosynthetic needs.

The present study is the first to examine in detail thefunctioning of the TCA cycle in H. pylori. It attempted to obtaina better understanding of the physiology of the bacterium and toaid in the identification of potential targets for therapeutic

Correspondence to G. Mendz, School of Biochemistry and Molecular

Genetics, The University of New South Wales, Sydney, NSW 2052,

Australia. Fax: +61 2 9385 1483, Tel: +61 2 9385 2042,

E-mail: [email protected]

Abbreviations: GABA, g-aminobutyrate

Enzymes: citrate synthase (EC 4.1.3.7); fumarate reductase (EC 1.3.99.1);

isocitrate dehydrogenase (EC 1.1.1.41); isocitrate lyase (EC 4.1.3.1);

a-ketoglutarate dehydrogenase (EC 1.2.4.2); a-ketoglutarate oxidase; malate

dehydrogenase (EC 1.1.1.37); malate synthase (EC 4.1.3.2); succinate

dehydrogenase (EC 1.3.99.1); succinyl-CoA synthetase (EC 6.2.1.5)

(Received 14 September 1998, revised 26 November 1998, accepted 30

November 1998)

q FEBS 1999 Tricarboxylic acid cycle of Helicobacter pylori (Eur. J. Biochem. 260) 259

intervention. Enzyme activities were examined to elucidate theirfunctional characteristics, which depend on both their intrinsicphysicochemical properties and their interactions with othercellular components. It is expected that the basic mechanisms ofenzymes will be the same in situ and in cell extracts as inpurified proteins, but modulation of their activities may dependcritically on the cellular milieu, thus in situ studies may revealproperties which otherwise could remain undetected.

MATERIALS AND METHODS

Oxaloacetate, cis-aconitate, trans-aconitate, succinate, fumarate,isocitrate, a-ketoglutarate, coenzyme A, BSA fraction V, poly-myxin B and trimethoprim were obtained from Sigma (St.Louis, MO, USA). Vancomycin and amphotericin B were fromSquibb (Princeton, NJ, USA) and Eli Lilly (West Ryde, NSW,Australia), respectively. NAD+, NADP+, NADH and NADPHand acetyl-CoA were from Boehringer Mannheim (North Ryde,NSW, Australia). All other reagents were of analytical grade.

Culture conditions and preparation of cell lysates

H. pylori NCTC 11639 was grown on Blood Agar Base no. 2(Oxoid, Basingstoke, UK) plates supplemented with 5% (v/v)horse blood, polymyxin B (1.25 U´L21), trimethoprim (5mg´L21), vancomycin (10 mg´L21) and fungizone (2 mg´L21).Cultures were passaged every 24±36 h and incubated in a FormaStericult incubator (Marietta, OH, USA) in an atmosphere of10% CO2 in air, 95% humidity at 37 8C.

Log phase H. pylori cells (<24 h) were harvested in sterileNaCl (0.9% w/v), checked for purity under phase-contrastmicroscopy and tested for positive urease and catalase activity.Cells were washed three times by centrifugation at 17 000 g(4 8C, 5 min) and resuspended in sterile NaCl (0.9% w/v).Following the final wash, packed cells were resuspended to aconcentration of <108 cells´mL21 in sterile, degassed NaCl(0.9% w/v) containing 1 mm dithiothreitol. Cell lysates wereprepared by twice freeze-thawing cell suspensions in liquidnitrogen; phase-contrast microscopy was employed to verifygreater than 99% cell lysis.

Lysates prepared from cultures of Acinetobacter lwoffii andEscherichia coli strain K12 were used as positive controls forenzymes not found in H. pylori. A. lwoffii was grown on achemically defined liquid medium described by Beacham et al.[29]. Cultures were incubated at 24 8C in an orbital shakingincubator (180 r.p.m.) for 24 h and harvested by centrifugationat 4 8C (6000 g, 30 min). Cells were then washed twice with50 mm Tris/HCl buffer (pH 7.5) and finally resuspended in thisbuffer before being disrupted by sonication (Branson Ultra-sonics, Danbury, CT, USA) consisting of three cycles of 20 sultrasonic pulses followed by 1 min cooling. E. coli was grownaerobically in Luria Bertani broth medium [30] at 37 8C withshaking (180 r.p.m.). Anaerobic cultures of the same organismwere grown on plates of Luria Bertani agar in an anaerobic jar.Both aerobic and anaerobic cultures were incubated for 16 h andharvested by centrifugation at 4 8C (17 000 g, 5 min) in sterileNaCl (0.9% w/v), washed twice with sterile NaCl (0.9% w/v)and finally resuspended in sterile KCl (150 mm). Lysates wereprepared from both cultures by disrupting cells by sonication asdescribed above.

Enzyme assays

Determination of in situ enzyme activities were carried outeither spectrophotometrically or by NMR spectroscopy. Assays

were performed at 37 8C and pH 7.4 with either 50 mm potas-sium phosphate or Hepes/KOH buffer under both aerobic andanaerobic conditions, with the latter achieved by either bubblingN2 through all buffers and substrate solutions or by the additionof sodium dithionite. Enzyme activities were reported as nmol´min21´mg21 protein, with protein concentrations of the extractsdetermined by the bicinchoninic acid method employing amicrotitre protocol (Pierce Chemical Co., Rockford, IL, USA)with BSA as standard. All values given are the result of at leastduplicate determinations on two independent experiments.

Citrate synthase activity was assayed by [1H]-NMR spectro-scopy using acetyl-CoA and oxaloacetate as substrates. Assaysfor inhibition of citrate synthase activity were carried out with2.0 mm acetyl-CoA and oxaloacetate in 50 mm phosphate buffer(pH 7.4). No additional salts were added as they appear todesensitize other citrate synthases to inhibition by some allostericinhibitors [31]. Aconitase activity was assayed by either [1H] or[13C]-NMR using either cis-aconitate or isocitrate as substrate.Isocitrate dehydrogenase activity was assayed spectrophoto-metrically [32] and by [1H]-NMR spectroscopy under the sameconditions by following the disappearance of isocitrate andappearance of a-ketoglutarate resonances. a-Ketoglutarate dehy-drogenase activity was assayed spectrophotometrically [32] withfurther analyses being performed using [1H]-NMR spectroscopywith the same assay conditions. a-Ketoglutarate oxidase activitywas assayed by [1H]-NMR spectroscopy using a-ketoglutarateas substrate and either benzyl viologen, FAD+ or NAD+ as theelectron acceptor by following the disappearance of a-keto-glutarate and appearance of succinate resonances. Succinatedehydrogenase activity was assayed spectrophotometrically bythe method of Veeger et al. [33] and by [1H]-NMR spectroscopyby following the disappearance of succinate in assay mixtureswith 50 mm succinate and 10 mm of either benzyl viologen orFAD+ as the electron acceptor. Fumarate reductase and fumaraseactivities were assayed by [1H]-NMR spectroscopy as pre-viously described [23]. Fumarase activity was also assayedspectrophotometrically according to Reeves et al. [32]. Succi-nyl-CoA synthetase activity was determined by both thespectrophotometric assays of Bridger et al. [34] and Reeveset al. [32] and by [1H]-NMR spectroscopy measuring thedisappearance of succinate and formation of succinyl-CoAfrom initial mixtures of 2±50 mm succinate, 20 mm CoA-SHand 10 mm ATP (or GTP). Malate dehydrogenase activity wasassayed spectrophotometrically [35]. Malate dehydrogenasekinetics with malate and NAD+ were performed after a shortpreincubation with malate to allow the malate/fumarate equi-librium (catalyzed by fumarase) to be established. An equi-librium constant of 3.15:1 in favour of malate was determinedfor H. pylori fumarase by [1H]-NMR spectroscopy and used forcalculating the initial malate concentration prior to NAD+

addition. Malate dehydrogenase activity was also analysed with[1H]-NMR spectroscopy by following the appearance or dis-appearance of malate and oxaloacetate. Isocitrate lyase activitywas assayed spectrophotometrically [32] and by [1H]-NMRspectroscopy by following the disappearance of isocitrate andappearance of succinate and glyoxylate. Malate synthase activitywas measured using spectrophotometry [32] and [1H]-NMRspectroscopy by following the disappearance of acetyl-CoA andglyoxylate and appearance of malate (and fumarate because ofthe presence of fumarase) resonances. The presence of the g-aminobutyrate (GABA) shunt was determined spectrophoto-metrically by assaying for GABA transaminase and succinicsemialdehyde dehydrogenase according to Bergmeyer et al.[35]. [1H]-NMR spectroscopic analysis of these enzymes wasalso performed using 10 mm GABA, a-ketoglutarate and

260 S. Pitson et al. (Eur. J. Biochem. 260) q FEBS 1999

NADP+ as substrates for GABA transaminase, and 10 mm suc-cinic semialdehyde and NADP+ (or NAD+) as substrates forsuccinate semialdehyde dehydrogenase.

The validity of all NMR spectroscopic enzyme assays wasdetermined by: (a) extended incubation of the reaction mixturesin the absence of enzyme extracts to account for any chemicalreactions, and (b) omission of each of the essential substrates orcoenzymes sequentially from the complete reaction mixture(containing bacterial extract) to determine nonspecific enzymereactions. Any reactions observed during this validation proce-dure are noted in the Results.

Nuclear magnetic resonance spectroscopy

For [1H]-NMR spectroscopy, bacterial lysates prepared in NaClwere placed into 5-mm tubes (Wilmad, Buena, NJ, USA),equilibrated at 37 8C, and the appropriate substrates added tostart the reactions. Free induction decays were collected using aBruker DMX-500 spectrometer (Karlsruhe, Germany), operat-ing in the pulsed Fourier transform mode with quadraturedetection. One-dimensional proton spectra were acquired at37 8C with presaturation of the water resonance. The instru-mental parameters were: spectral width 5434.78 Hz, memorysize 8 K, acquisition time 0.754 s, number of transients 64±128,and relaxation delays with solvent presaturation 1.253±2.745 s.Exponential filtering of 0.5 Hz was applied prior to Fouriertransformation. Chemical shifts are quoted relative to externalsodium 4,4-dimethyl-4-silapentane-1-sulfonate at 0 ppm. [13C]-NMR spectra were acquired on a Bruker ACP-300 spectrometeroperating at 37 8C in the pulsed Fourier transform mode withquadrature detection and composite pulse decoupling of protons.The instrumental parameters for observing [13C]-NMR were:operating frequency 75.47 MHz, spectral width 16129 Hz,memory size 16 K, acquisition time 0.508 s, pulse angle 608(9 ms), number of transients 1200, and relaxation delay 0.309 s.Exponential filtering of 1 Hz was applied prior to Fouriertransformation.

The time-evolution of the substrate and products was fol-lowed by acquiring sequential spectra of the reactions. Progresscurves were obtained by measuring the integrals of substrate andproduct resonances at each point in time. Maximal rates werecalculated from good fits (correlation coefficients $ 0.99) ofthe data to straight lines for 30±120 min of the incubations.Calibrations of the peaks arising from substrates were performedby extrapolating the resonance intensity data to zero time andassigning to this intensity the appropriate concentration value.The intensity of resonances corresponding to products wascalibrated by adding the appropriate metabolite to cell suspen-sions and constructing standard concentration curves.

Kinetic parameters

Kinetic parameters were determined from the initial rates oftime-course measurements for at least eight concentrations ofeach substrate. The data were then analysed by nonlinearregression analysis using the program EnzymeKinetics (TrinitySoftware, Campton, NH, USA).

Cellular location of the enzymes

To ascertain whether the enzymes were located in the cytosol orassociated with the cell envelope, H. pylori lysates were sepa-rated into pellet (cell envelope) and supernatant (cytosol)fractions by centrifugation at 20 000 g for 10 min at 4 8C.The pellet was then washed three times with 150 mm KCl. This

is a gentle fractionation method in which components associatedwith the cell wall remain in the pellet which is composed almostexclusively of intact cell envelopes [18].

H. pylori genome analysis

Putative TCA cycle enzymes from the H. pylori completegenome [9] were searched against a nonredundant amino acidsequence database at the Australian National Genome Informa-tion Service (ANGIS) using BLASTP [36]. Percentage identitiesof aligned sequences were then determined using GAP [37].

RESULTS

Citrate synthase activity

Citrate synthase catalyses the aldol condensation of acetyl-CoAand oxaloacetate to yield citrate and CoA-SH. A [1H]-NMRmethod was used to assay this enzyme activity in H. pylorilysates (Fig. 1) because the common spectrophotometric assayof Srere et al. [38] using the chromogenic reagent 5,5 0-dithiobis-(2-nitrobenzoic acid) (DTNB) could not be employedowing to strong `DTNB reductase' activity present in theH. pylori extracts [39]. The presence of an acetyl-CoA hydro-lase (EC 3.1.2.1) with a specific activity of 24.2 nmol´min21´mg21 was measured in H. pylori extracts and takeninto account during the citrate synthase assays.

H. pylori citrate synthase activity was located in the cytosol,

Fig. 1. Aliphatic region of the [1H]-NMR spectra of a time-course of

citrate synthase activity in H. pylori strain NCTC 11639 extracts

incubated at 37 8C. Substrate concentrations were 5 mm. The resonances

corresponding to the substrates oxaloacetate and acetyl-CoA, and the

product citrate are indicated on the figure. Protein concentration was

0.489 mg´mL±1. The time at which each spectrum was acquired is shown on

the left-hand side.


with only 13% activity found to be associated with the cellenvelope. Kinetic analyses yielded Km values of 204 ^ 34 mmfor acetyl-CoA and 347 ^ 43 mm for oxaloacetate, at saturatingconcentrations of the other substrate, and a Vmax of 169 ^11 nmol´min21´mg21 (Table 1).

To characterize the H. pylori citrate synthase activity, theeffects of a number of metabolites were examined and theenzyme activity classified based on these effects [31,40].Although NADH inhibits the citrate synthases of most Gramnegative bacteria [40], the coenzyme did not inhibit H. pyloricitrate synthase activity at concentrations of up to 20 mm; thislack of effect is a characteristic property of citrate synthasesfrom gram-positive bacteria and eukaryotes [40]. The effects ofATP, ADP and AMP on H. pylori citrate synthase activity werealso examined as these nucleotides are known to inhibit mostcitrate synthases from microbial, plant and animal sources [40].ADP or AMP did not affect H. pylori enzyme activity, but ATPinhibited it competitively with respect to acetyl-CoA with a Ki of13.0 ^ 1.5 mm. Unlike some other citrate synthases [40] theobserved inhibition of the H. pylori activity by ATP was notrelieved by the presence of Mg2+ ions (10 mm MgCl2). a-Ketoglutarate and succinyl-CoA also have been reported toinhibit bacterial citrate synthase activities [31,40], but neither ofthese metabolites at 10 mm concentration had detectable effectson citrate synthase activity in H. pylori.

Aconitase activity

Aconitase catalyzes the equilibrium isomerization of citrate toisocitrate via cis-aconitate. [1H]- or [13C]-NMR spectroscopywere employed to measure this activity in situ in H. pylori, byincubating isocitrate or cis-aconitate with bacterial lysates andobserving the disappearance of these substrates and the appear-ance of citrate (Fig. 2).

Kinetic determinations with cis-aconitate as substrate yieldeda Km of 6.8 ^ 0.8 mm and a Vmax of 301 ^ 12 nmol´min21

mg21. The activity appeared oxygen-stable, with no differencesin aconitase activity observed between aerobic or anaerobicassay conditions, or in lysates prepared and measured with orwithout 1 mm dithiothreitol (Table 1).

Fig. 2. Aliphatic region of the [13C]-NMR spectra of a time-course of

aconitase activity in H. pylori strain NCTC 11639 extracts incubated at

37 8C. Substrate concentrations were 50 mm. The resonances corresponding

to the substrate cis-aconitate, and the products citrate and isocitrate are

indicated on the figure. The time at which each spectrum was acquired is

shown on the left-hand side. The insert shows the nonlinear regression

analysis of aconitase kinetic data.

Table 1. Summary of kinetic parameters and cellular location of TCA cycle enzyme activities of H. pylori.

Enzyme Substrate

Km

(mm)

Vmax

(nmol´min±1mg±1) Location

Citrate synthase Acetyl-CoA

Oxaloacetate

0�.204

0.347

169 Cytosol

Aconitase cis-Aconitate 6�.8 301 Cytosol

a-Ketoglutarate oxidase a-Ketoglutarate

FAD+

7�.4

1.4

15�.8 Cytosol

a-Ketoglutarate oxidase a-Ketoglutarate 26�.3 54�.8 Cytosol

(with 2 mm CoA-SH) FAD+ 28�.7

Fumarate reductase Fumarate 0�.830 2400 Envelope

Fumarase Fumarate

Malate

121�

7.3

4100

10800

Cytosol

Malate dehydrogenase Malate 0�.177 40 Cytosol

(oxidative direction) NAD+ 0�.161

Malate dehydrogenase Oxaloacetate 0�.128 111 Cytosol

(reductive direction) NADH 0�.065

Malate synthase Glyoxylate

Acetyl-CoA

8�.5

3.2

91 Cytosol


Relative to the activity in whole lysates, only 45 ^ 8% ofaconitase activity was observed in the cytosol after lysate frac-tionation, and no activity was detected in the cell envelopefraction. This loss of aconitase activity following fractionationwas investigated by comparing the activities in lysates withthose of reconstituted lysates obtained by recombination of cellenvelope and cytosolic fractions after centrifugation. Full aconi-tase activity was measured in recombined lysates. However,adding KCl (150 mm) to the lysates before fractionation,resulted in recovery of only <50 ^ 7% of the original activityafter reconstitution of the two fractions. Aconitase activity in thelysates or cytosol was unaffected by 150 mm KCl. This sug-gested that although aconitase activity appeared to be cytosolic,interactions with the cell envelope were required for full enzymeactivity.

Isocitrate dehydrogenase activity

Isocitrate dehydrogenase catalyzes the oxidative decarboxylationof isocitrate to a-ketoglutarate. In H. pylori isocitrate dehydro-genase was located primarily (. 96%) in the cytosolic fraction,and like in most bacteria [40] was NADP+ specific, showing nodetectable activity with NAD+. The activity was inhibited athigher concentrations of both NADP+ and isocitrate, and sig-moidal kinetics were observed for both these substrates sug-gesting that the enzyme was under allosteric regulation. Anoptimal isocitrate dehydrogenase activity of 245 nmol´min21

mg21 was observed in H. pylori cytosolic extracts with 0.3 mmisocitrate and 0.5 mm NADP+.

Isocitrate dehydrogenase, like citrate synthase, is consideredan important regulatory enzyme in the TCA cycle of manyorganisms [41,42]. In order to investigate its role in H. pylori theeffects of a number of potential effectors on its activity wereexamined. Similar to other bacterial isocitrate dehydrogenases[40], a slight stimulation (10%) of activity in H. pylori wasobserved in the presence of 0.5 mm AMP. However, no effectswere detected with the same concentrations of ADP or ATP, orwith 5 mm of glyoxylate or pyruvate, which are known to affectisocitrate dehydrogenase activities from some other organisms[31,40].

a-Ketoglutarate dehydrogenase and a-ketoglutarate oxidaseactivities

a-Ketoglutarate dehydrogenase catalyses the oxidative decar-boxylation of a-ketoglutarate to succinyl-CoA. No activity wasdetected in H. pylori lysates using either spectrophotometric or[1H]-NMR methods which showed activity in cell-free extractsof A. lwoffii and E. coli. This lack of a-ketoglutarate dehy-drogenase activity in H. pylori was in agreement with studies byHoffman et al. [27] and Corthesy-Theulaz et al. [25].

[1H]-NMR spectroscopy revealed the direct oxidation of a-ketoglutarate to succinate when FAD+ or benzyl viologen wassupplied as the electron acceptor. No formation of succinyl-CoAwas observed in the reaction at any stage. Analysis of succinyl-CoA hydrolysis indicated only low succinyl-CoA hydrolaseactivity (2.1 nmol´min21mg21), which would not account forthe absence of this compound if it had been formed during theoxidation of a-ketoglutarate. The result suggested the presenceof a-ketoglutarate oxidase activity, which was confirmed by theobservation that CoA-SH was not required for the oxidation ofa-ketoglutarate to succinate.a-ketoglutarate oxidase activity was found entirely in the

cytosolic fraction. At saturating concentrations of the othersubstrate, Km values of 7.4 ^ 0.7 mm and 1.38 ^ 0.09 mm

were determined for a-ketoglutarate and FAD+, respectively,and a Vmax of 15.8 ^ 0.3 nmol´min21mg21 (Table 1). SimilarKm and Vmax values were obtained when benzyl viologen wasemployed as the electron acceptor instead of FAD+. No activitywas observed when NAD+ or NADP+ were supplied as electronacceptors.

Endogenous CoA-SH concentrations in the samples wereestimated to be negligible. Addition of CoA-SH was notrequired for the reaction catalyzed by a-ketoglutarate oxidase,but its presence resulted in a marked increase in enzyme activity.A dose±response plot for CoA-SH gave a sigmoidal curve(Fig. 3) suggesting an allosteric interaction with the enzyme.Kinetic determinations for a-ketoglutarate oxidase activity inthe presence of 2 mm CoA-SH gave Km values of 26.3 ^2.8 mm and 28.7 ^ 2.67 mm for a-ketoglutarate and FAD+,respectively, at saturating concentrations of the other substrate,and a Vmax of 54.8 ^ 4.2 nmol´min21mg21 (Table 1).

a-Ketoglutarate oxidase activity was enhanced under anae-robic conditions created by bubbling N2 through all buffers andsubstrate solutions, but more than 70% activity was measuredwhen these conditions were not established. A probable reasonis that the environment in the NMR assays is essentially micro-aerophilic. It should be noted that the spectrophotometric assaydescribed by Hoffman et al. [27] in which the reaction isfollowed by the reduction of benzyl viologen at 546 nm afteraddition of sodium dithionite to render the conditions anaerobic,could not be employed because sodium dithionite reducesbenzyl viologen [43], and gave very inconsistent results withenzyme assays.

Succinyl-CoA synthetase activity

The only energy-yielding substrate-level phosphorylation thattakes place in the TCA cycle is the catalysis by succinyl-CoAsynthetase of succinyl-CoA to succinate and CoA-SH with theformation of a nucleotide triphosphate. Under the experimentalconditions employed, no succinyl-CoA synthetase activity wasobserved in H. pylori lysates with either spectrophotometric[34] or [1H]-NMR spectroscopic methods. Lysates of E. coligrown under aerobic conditions gave detectable activities inthese assays, and thus act as positive controls.

In should be noted that the spectrophotometric assay of

Fig. 3. Effect of CoA-SH concentration on a-ketoglutarate oxidase

activity in H. pylori strain NCTC 11639 extracts. The data show a

sigmoidal function characteristic of allosteric effects.


Reeves et al. [32] based on detecting succinohydroxamic acidcomplexes could not be used for H. pylori extracts owing tostrong background reactions involving the glutathione in theassay mixture. Similar problems were recently reported for thisassay with H. pylori by Corthesy-Theulaz et al. [25].

Succinate dehydrogenase activity

Succinate dehydrogenase catalyses the reversible dehydrogena-tion of succinate to fumarate. Under the experimental conditionsemployed no succinate dehydrogenase activity was observed inH. pylori lysates with either spectrophotometric or [1H]-NMRspectroscopy assays employing benzyl viologen, FAD+, NAD+

or NADP+ as electron acceptors. Lysates of E. coli grownaerobically yielded detectable activity under the same assayconditions using NAD+ as electron acceptor.

Fumarase and fumarate reductase activities

Fumarase catalyses the reversible hydration of fumarate to formmalate, while fumarate reductase catalyses the unidirectionalconversion of fumarate to succinate. The presence and someproperties of these enzyme activities in H. pylori have beenpreviously examined by [1H]-NMR spectroscopy [15,18]. Fuma-rase activity was found primarily (95%) in the cytosol. Thekinetic parameters were a Km and Vmax for fumarate of 121 ^12 mm and 4.1 ^ 0.2 mmol´min21mg21, respectively, and a Km

and Vmax for malate of 7.3 ^ 0.4 mm and 10.8 ^ 0.2 mmol´min21mg21, respectively (Table 1). H. pylori fumarate reduc-tase activity was found entirely in the cell envelope fraction asfor most other bacteria [44]. Fumarate reductase activity wassubstrate inhibited at high fumarate concentrations (20 mm) andKm and Vmax values of 0.83 ^ 0.06 mm and 2.40 ^ 0.06 mmol´min21mg21, respectively, have been previously obtained [18](Table 1). When fumarate was supplied to whole cell or lysatesuspensions the reaction proceeded to completion in thedirection of succinate formation [18].

Malate dehydrogenase activity

Malate dehydrogenase catalyses the reversible dehydrogenationof malate to oxaloacetate, coupled with the reduction of NAD+

to NADH. H. pylori malate dehydrogenase activity was foundonly in the cytosolic fraction. Spectrophotometric assays of thereaction in the direction of malate production yielded Km valuesof 128 ^ 16 mm and 65 ^ 5 mm for oxaloacetate and NADH,respectively, at saturating concentrations of the other substrate,and a Vmax of 111 ^ 3 nmol´min21mg21. In the oxidativedirection, malate dehydrogenase activity gave Km values of177 ^ 15 mm and 161 ^ 24 mm for malate and NAD+, respect-ively, and a Vmax of 40 ^ 2 nmol´min21mg21 (Table 1).

Enzyme activities of the glyoxylate bypass

The presence of the glyoxylate bypass in H. pylori was inves-tigated by searching for isocitrate lyase and malate synthaseactivities. Isocitrate lyase activity was not detected in H. pyloriunder the experimental conditions used, employing either spec-trophotometric or [1H]-NMR methods which showed activity inE. coli lysates employed as controls.

Malate synthase activity was detected by [1H]-NMR spectro-scopy (Fig. 4), and more than 98% was present in the cytosolicfraction. Kinetic analysis of malate synthase activity gave Km

values of 8.5 ^ 1.1 mm and 3.2 ^ 0.4 mm for glyoxylate andacetyl-CoA, respectively, at saturating concentrations of the

other substrate; and a Vmax of 91 ^ 4 nmol´min21mg21

(Table 1).

Enzyme activities of the g-aminobutyrate shunt

The GABA shunt is an alternative metabolic pathway thatconverts a-ketoglutarate to succinate in the absence of a-ketoglutarate dehydrogenase. The GABA shunt involves severalenzymes, with the pathway proceeding via glutamate, GABAand succinate semialdehyde through the action of glutamatedehydrogenase (EC 1.4.1.2 and EC 1.4.1.4), glutamate decar-boxylase (EC 4.1.1.15), GABA transaminase and succinicsemialdehyde dehydrogenase. Under the experimental condi-tions used no GABA transaminase or succinic semialdehydedehydrogenase activity was detected in H. pylori lysates,employing either spectrophotometry or [1H]-NMR spectroscopy,suggesting the GABA shunt is not operational in H. pylori.

H. pylori genome and the TCA cycle

The following detailed analysis of the H. pylori proteinsidentified as being TCA cycle enzymes is based on the generalanalysis of the H. pylori genome performed by Tomb et al. [9].

The H. pylori citrate synthase amino acid sequence has 47.8%similarity with the Rickettsia prowazekii enzyme, and has beenfound to have a similar homology with citrate synthases from alarge number of other organisms, including several otherRickettsia species (46±47%), Pseudomonas aeruginosa (46.1%),Acinetobacter antiratum (45.3%), Rhizobium tropici (45.3%),Acetobacter aceti (44.5%) and E. coli (43.1%). H. pylori citratesynthase has much lower identity with porcine heart citratesynthase (26.3%), the only citrate synthase for which thecomplete 3-dimensional structure is known. Our analyses

Fig. 4. Aliphatic region of the [1H]-NMR spectra of a time-course of

malate synthase activity in H. pylori strain NCTC 11639 extracts

incubated at 37 8C. Substrate concentrations were 5 mm. The resonances

corresponding to the substrates glyoxylate and acetyl-CoA and the products

malate and fumarate are indicated on the figure. The time at which each

spectrum was acquired is shown on the left-hand side.


indicate that all seven oxaloacetate/citrate binding site residuesin porcine citrate synthase [45,46] are conserved in the H. pylorienzyme. Similar conservation of oxaloacetate/citrate bindingsite residues is present in the E. coli [47] and R. prowazekiicitrate synthase [48]. Greater variation was found in the residuesof the acetyl-CoA binding site, with only one of the four bindingsites of the porcine citrate synthase [45,46] showing similaritywith the residues of the H. pylori enzyme. The H. pylori citratesynthase amino acid sequence also differed substantially fromthe P. aeruginosa and E. coli sequences in the region involved inthe allosteric binding of NADH in these enzymes (Cys206 [49]).

H. pylori aconitase shows 64% amino acid sequence identitywith E. coli aconitase B, indicating that both enzymes representa distinct class of aconitase with amino acid sequences differentfrom other aconitases so far described, with the possible excep-tion of the Neisseria gonorrhoeae aconitase for which only apartial sequence has been determined [50]. Even after the fourdomains of the H. pylori aconitase were rearranged in a 4±1±2±3 fashion [50,51], they showed only 18% amino acid sequenceidentity with porcine mitochondrial aconitase. However, in thisconfiguration, 16 of the 20 catalytic residues were conservedbetween these two enzymes, including the three cysteinylsinvolved in ligand binding in the iron-sulphur centre. Similarresults were also observed by Bradbury et al. [51] duringcomparison of porcine aconitase and E. coli aconitase B.

Tomb et al. [9] reported H. pylori isocitrate dehydrogenase tohave a 70.7% amino acid sequence identity with the E. colienzyme, but our analyses showed slightly higher identities withisocitrate dehydrogenase from Salmonella enterica (71.8%) andCitrobacter diversus (71.6%). Lower identities were observedwith isocitrate dehydrogenases from Bacillus israeli (66.8%),Vibrio spp. (66.3%), Bacillus subtilis (66.0%), and Thermusaquaticus (65.9%). Further sequence comparisons with E. coliisocitrate dehydrogenase, the most extensively studied of theseenzymes, indicated that the residues critical for substrate andcofactor binding, catalysis or involved in conformationalchanges in enzyme configuration in this enzyme [52,53], wereconserved in H. pylori isocitrate dehydrogenase, includingSer113, a residue that is phosphorylated during E. coli isocitratedehydrogenase inactivation catalysed by isocitrate dehydrogen-ase phosphatase/kinase [54,55]. The results for the H. pylorienzyme were in agreement with the general observation thatmany amino acids are conserved among isocitrate dehydro-genases from very distantly related organisms [56].

Tomb et al. [9] reported that H. pylori fumarase has highestamino acid sequence identity with E. coli fumarase C (63.7%),but our analysis showed greater identity with B. subtilis fuma-rase (66.7%). Lower identities were found for the fumarasesfrom Haemophilus influenzae (61.3%), Rhizopus oryzae (60.8%),Myxococcus xanthus (59.4%) and Bradyrhizobium japonicum(56.3%). Detailed sequence comparison with the E. coli fuma-rase, the most extensively studied prokaryotic fumarase, indi-cated that the three regions comprising segments 129±146,181±200 and 312±331 [57], that constitute the active site[58,59], and are conserved in many fumarases, also were highlyconserved in H. pylori fumarase (96.6% identity).

H. pylori fumarate reductase subunits A, B and C showedamino acid sequence identities of 69.5%, 69.9% and 56.3% withthe three Wolinella succinogenes fumarate reductase subunits A,B and C, respectively. This result supports the ribosomal RNAsequence analyses that indicate that W. succinogenes is closelyrelated to H. pylori [60]. Further analysis of these sequencesindicated that the 11 conserved regions identified in W. succino-genes fumarate reductase A and related enzymes [61], were alsoconserved in H. pylori fumarate reductase A (91.5% identity),

particularly the regions thought to involve FAD+ and fumaratebinding (93.5% identity) [61]. The three cysteine clusters iden-tified in W. succinogenes fumarate reductase B [61], andprobably associated with ligand binding in the iron-sulphurcentres, also were conserved fully in H. pylori fumaratereductase B.

Finally, activities corresponding to several enzymes, includ-ing malate dehydrogenase, malate synthase and a-ketoglutarateoxidase not identified in the H. pylori genome were detected inthis study. Other enzyme activities, including a-ketoglutaratedehydrogenase, succinyl-CoA synthetase, succinate dehydro-genase, isocitrate lyase, GABA transaminase and succinicsemialdehyde dehydrogenase were not detected in H. pylori;and sequences known to encode enzymes of these activities werenot found by genomic analysis.

DISCUSSION

In many organisms the flux of metabolites through the TCAcycle proceeds in the oxidative direction from citrate to oxalo-acetate, but several patterns of the cycle have been identified indifferent organisms. For example, when E. coli is grown underanaerobic conditions with glucose as the carbon source, a-ketoglutarate dehydrogenase is not expressed and the TCA cyclebecomes a noncyclic branched pathway for production of suc-cinate and succinyl-CoA on one branch, and a-ketoglutarate onthe other [62]. Growth of E. coli under these conditions alsoresults in much lower levels of all TCA cycle enzymes and thederepression of fumarate reductase, resulting in enhanced suc-cinate and succinyl-CoA formation in the reductive branch.Similar branched TCA cycles have been reported in a number ofanaerobic organisms [31]. Moreover, a-ketoglutarate dehydro-genase is absent in cyanobacteria and the resultant incompletecycle does not appear geared toward metabolic energy produc-tion, but instead operates in conjunction with a glyoxylatebypass so that isocitrate is either decarboxylated to a-keto-glutarate or converted to succinate and malate [31]. The gly-oxylate bypass and the complete oxidative TCA cycle operateconcurrently in E. coli when acetate is the sole source of carbonand metabolic energy to sustain aerobic growth. A junction iscreated at isocitrate where isocitrate lyase and isocitrate dehy-drogenase compete for the common substrate, with the fluxthrough the glyoxylate bypass generating precursors used forbiosynthesis, whilst most of the flux through the TCA cycle isdevoted to the supply of reducing power and ATP [41].

In the current study the properties of TCA cycle enzymeactivities of the microaerophilic gastric pathogen H. pylori weredetermined using in situ analyses to obtain information onenzyme function in an environment closer to that present in thecellular milieu. [1H]- and [13C]-NMR spectroscopy was employedto overcome the difficulties in the measurement of these enzymeactivities using spectrophotometric assays in in situ studies, andhelped to provide the first complete picture of the H. pyloriTCA cycle. The use of NMR spectroscopy to follow enzymereactions represented a major advantage over the often lessspecific spectrophotometric assays employed previously tostudy bacterial TCA cycles in situ, because in most cases bothsubstrate degradation and product formation can be followeddirectly using appropriate NMR techniques. Examples of theadvantages of NMR spectroscopy methods were clearly shownby comparing the results obtained for the measurements ofcitrate synthase, a-ketoglutarate oxidase and succinyl-CoA syn-thetase enzyme activities. This appeared to be a major reason forthe differences between the results of this investigation andthose reported by Hoffman et al. [27], which reported the


presence of an intact, complete oxidative TCA cycle andglyoxylate bypass in H. pylori.

The kinetic properties of the H. pylori TCA cycle enzymeswere somewhat unusual, with much higher Km values (for mostof the enzymes) than those observed for other bacteria (Table 1).This would suggest that the TCA cycle in H. pylori is onlysubstantially operational during growth in nutrient-rich environ-ments where metabolites are plentiful.

It has been shown that the H. pylori TCA cycle is a noncyclic,branched pathway that appears directed towards the productionof biosynthetic intermediates more than `metabolic energy'[28,63], with the production of succinate in the reductive branchand a-ketoglutarate in the oxidative branch (Fig. 5). Thisbranched `cycle' is similar to the pathway found in anaerobicmetabolism, a conclusion supported by the presence of fumaratereductase and absence of succinate dehydrogenase activities.The oxidative and reductive branches are metabolically linkedby the presence of a-ketoglutarate oxidase activity, an enzymeactivity which should not be considered a proper part of theTCA cycle. No other pathways linking the tricarboxylic acid anddicarboxylic acid branches, such as the glyoxylate bypass or theGABA shunt, were detected under the growth conditionsemployed, in contrast to observations in other microaerophilicbacteria including Rhizobium and Bradyrhizobium species [64,65].

Several enzymes of the H. pylori TCA cycle showed unusualproperties in this study. Unlike most gram-negative bacterialcitrate synthases, the activity of the H. pylori enzyme wasunaffected by NADH and a-ketoglutarate, but was inhibited byATP and acetyl-CoA. Extensive studies of the subunit structure,catalytic activity and regulatory properties of citrate synthase[31,40] have shown that, generally, eukaryotic and gram-positive bacterial enzymes are dimeric and show allostericregulation with ATP, while gram-negative bacterial citrate syn-thases are hexameric and show allosteric regulation with NADHand a-ketoglutarate. Thus, although H. pylori is a gram-negativebacterium, its citrate synthase activity has more in common withthe regulatory properties of eukaryotes and gram-positive bacteria.This finding is unusual, but not unique, because there are severalother gram-negative citrate synthases with similar regulatoryproperties, including those from Acetobacter europaeus [66],Methylophilus methylotrophus (and other methylotrophs) [31],cyanobacteria [31], and also the citrate synthase from R. pro-wazekii [67] which showed the greatest amino acid sequencesimilarity to the H. pylori enzyme. Examination of the H. pyloricitrate synthase amino acid sequence showed substantial

differences in the region around the proposed allosteric bindingsite for NADH compared with sequences from E. coli andP. aeruginosa citrate synthase [68], which probably accounts forthe lack of NADH inhibition in the H. pylori enzyme. This lackof inhibition may be explained from a metabolic perspective. Ifthe H. pylori TCA was not utilized as an energy-generatingpathway, it would appear inappropriate for an `energy-signal'like NADH to exert specific regulatory control. In contrast,inhibition by ATP appears to be more general and is probablyrelated to its structural similarity to acetyl-CoA [31].

H. pylori aconitase shows high sequence similarity withE. coli aconitase B, and together they comprise a distinct classof aconitase [50]. E. coli produces two aconitases (A and B), butinterestingly, only aconitase B is expressed during growth inanaerobic conditions [51,69], although the reasons for thisdifferential expression are not yet known. While E. coli aconi-tase B appears very oxygen labile [51], the H. pylori aconitaseactivity appeared much more stable. This enhanced stabilitymay be a result of the gentle breakage of the cells by freeze-thawing, as some instability in aconitase activity was observedwhen H. pylori cells were disrupted by sonication [27].

In this investigation only NADP+-dependent isocitrate dehy-drogenase activity was found in H. pylori lysates, in contrast toan earlier report by Hoffman et al. [27]. The H. pylori enzymeactivity was controlled by allosteric regulation, with sigmoidalkinetics, with the presence of AMP resulting in some stimu-lation in activity. This was unusual as generally only NAD+-dependant isocitrate dehydrogenases appear subject to this typeof regulation [40]. The presence of a conserved phosphorylationsite between the H. pylori and E. coli isocitrate dehydrogenasemay indicate the potential for the H. pylori enzyme activity tobe regulated additionally by reversible inactivation catalysed byisocitrate dehydrogenase phosphatase/kinase [42]. However, theabsence of isocitrate lyase activity and the apparent lack of theglyoxylate bypass pathway in H. pylori, may suggest thatregulatory control at this point in the cycle is not essential, andthat the phosphorylation site may be a redundant evolutionaryremnant, particularly when in E. coli, isocitrate dehydrogenasephosphatase/kinase is part of the same operon as the glyoxylatebypass enzymes isocitrate lyase and malate synthase [42].

The presence in H. pylori of a-ketoglutarate oxidase activity,rather than a-ketoglutarate dehydrogenase activity is interestingin the light of the finding by Hughes et al. [17] that H. pyloriappears to possess a pyruvate:flavodoxin oxidoreductase ratherthan pyruvate dehydrogenase, because both a-ketoglutarate

Fig. 5. The TCA cycle and associated pathways

of H. pylori. The diagram shows a TCA branch

comprising citrate synthase, aconitase and isoci-

trate dehydrogenase. It also shows a dicarboxylic

acid branch comprising malate dehydrogenase,

fumarase and fumarate reductase.


dehydrogenase and pyruvate dehydrogenase are structurallyand functionally related [70]. Very few studies have examineda-ketoglutarate oxidase, although similar enzymes have beenreported from Thermococcus litoralis [71], Heliobacterium spp.[72], and possibly a number of other obligate anaerobes [73].However, the T. litoralis enzyme activity, the best studied of thisgroup, differs from the H. pylori a-ketoglutarate oxidase acti-vity because it has an absolute requirement for CoA-SH [71],and therefore is probably a different type of enzyme. TheHeliobacterium spp. enzyme has not been as extensivelystudied, but functionally it is more similar to the H. pyloria-ketoglutarate oxidase as CoA-SH does not appear essential,but does enhance its activity [72].

An unusual observation regarding the H. pylori TCA cycle isthe presence of malate synthase activity, although under thegrowth conditions employed the glyoxylate bypass was notoperational, indicated by the absence of isocitrate lyase activity.Malate synthase activity suggested a possible alternative func-tion for this enzyme in H. pylori as a direct entry point forglyoxylate into the TCA cycle or into the glyoxylate oxidizingcycle [41]. The lack of succinyl-CoA synthetase activity inH. pylori was in agreement with results from a recent study byCorthesy-Theulaz et al. [25], but it raised the question of thesource of succinyl-CoA to supply substrate for the recentlyisolated succinyl-CoA: acetoacetyl-CoA transferase [25]. Furtherinvestigations are needed to resolve this question. Finally, thefact that malate dehydrogenase, malate synthase and a-keto-glutarate oxidase activities were observed in H. pylori, but havenot been identified in the H. pylori genome clearly suggestedthat these enzymes are likely to have amino acid sequences quitedistinct from the others so far determined. This example shouldserve as an illustration of the dangers of predicting metabolicand functional characteristics of organisms based solely ongenomic analysis.

In conclusion, this study has elucidated for the first time thecomposition and properties of the H. pylori TCA cycle, pro-vided insights into the overall metabolism of H. pylori, andinformation that may assist in the development of new thera-peutic agents against this pathogenic organism.

ACKNOWLEDGEMENTS

This work was made possible by the support of the National Health and

Medical Research Council of Australia and the Australian Research Council.

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The tricarboxylic acid cycle of Helicobacter pylori

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