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. Author manuscript; available in PMC: 2017 Dec 22.
Published in final edited form as: Cell Chem Biol. 2016 Nov 17;23(12):1480–1489. doi: 10.1016/j.chembiol.2016.10.007

Unsaturated fatty acid synthesis in the gastric pathogen Helicobacter pylori proceeds via a backtracking mechanism

Hongkai Bi 1,2,*, Lei Zhu 3,5, Jia Jia 1,2, Liping Zeng 1,2, John E Cronan 3,4,6,*
PMCID: PMC5215899  NIHMSID: NIHMS830960  PMID: 27866909

SUMMARY

Helicobacter pylori is a Gram-negative bacterium that inhabits human upper gastrointestinal tract and the presence of this pathogen in the gut microbiome increases the risk of peptic ulcers and stomach cancer. H. pylori depends on unsaturated fatty acid (UFA) biosynthesis for maintaining membrane structure and function. Although some of the H. pylori enzymes involved in UFA biosynthesis are functionally homologous with the enzymes found in Escherichia coli, we show here that an enzyme HP0773, now annotated as FabX, uses an unprecedented a backtracking mechanism to not only dehydrogenate decanoyl-acyl carrier protein (ACP) in a reaction that parallels that of acyl-CoA dehydrogenase, the first enzyme of the fatty acid β-oxidation cycle, but additionally isomerizes trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, the key UFA synthetic intermediate. Thus, FabX reverses the normal fatty acid synthesis cycle in H. pylori at the C10 stage. Overall, this unusual FabX activity may offer a broader explanation for how many bacteria that lack the canonical pathway enzymes produce UFA-containing phospholipids.

eTOC

Bi et al., report a flavin enzyme that reverses the normal fatty acid synthetic pathway to introduce a cis double bond into a saturated acyl chain. The enzyme has both dehydrogenation and isomerase activities. This enzyme allows unsaturated fatty acid synthesis by a non-canonical pathway.

INTRODUCTION

Unsaturated fatty acid (UFA) biosynthesis is required to maintain normal membrane structure and function in many groups of bacteria able to grow under anaerobic conditions. In the paradigm type II fatty acid biosynthesis system, that of Escherichia coli, the key player in UFA synthesis is FabA, the β-hydroxydecanoyl-ACP dehydratase/isomerase discovered by Bloch and coworkers (Bloch, 1971), which introduces the UFA double bond into the growing acyl chain during elongation of octanoyl-ACP to decanoyl-ACP. FabA uses the same active site to catalyze both dehydration of β-hydroxydecanoyl-ACP to generate trans-2-decenoyl-ACP and the isomerization of this intermediate to cis-3-decenoyl-ACP (Bloch, 1971; Nguyen et al., 2014). Formation of cis-3-decenoyl-ACP diverts the nascent acyl chain into the UFA branch of the fatty acid synthetic pathway (Kass et al., 1967:Kass, 1967 #10). The cis-3-decenoyl-ACP intermediate is then elongated to the twelve carbon 3-oxo-5-dodecenoyl-ACP by FabB (3-oxoacyl-ACP synthase I), the other enzyme essential for E. coli UFA synthesis (Feng and Cronan, 2009). Note that FabZ, the enzyme responsible for the dehydration reaction of the fatty acid synthetic cycle, cannot catalyze the isomerization reaction.

Other bacteria have variations on the classical pathway. Some Gram-positive bacteria, such as Streptococci, utilize an isomerase, FabM, to convert trans-2-decenoyl-ACP to cis-3-decenoyl-ACP (Marrakchi et al., 2002). Aerococcus viridans has FabQ which replaces the function of E. coli FabZ in vivo and also catalyzes the isomerization required for UFA biosynthesis (Bi et al., 2013).

An outlier is an unidentified anaerobic UFA synthesis mechanism dependent on a gene called ufaA reported in Neisseria gonorrhoeae (Isabella and Clark, 2011b). UfaA homologues were suggested to be widespread in bacteria lacking FabA and FabM. In addition, the chemistry involved in UFA synthesis in anaerobic Neisseria was proposed to be distinct from that of the classical anaerobic FabA-FabB pathway.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium that colonizes the gastric mucosa of approximately half of the world population. H. pylori phospholipids have an unusually simple fatty acid composition in that tetradecanoic acid (C14:0) and the cyclopropane fatty acid, cis-11, 12-methyleneoctadecanoic acid (C19:0 cyclo) are the major fatty acids, whereas hexadecanoic acid (C16:0), octadecanoic acid (C18:0) and octadecenoic acid (C18:1) are only trace components (Geis et al., 1990). The H. pylori genome contains genes that encode homologues of all E. coli enzymes required for saturated fatty acid (SFA) synthesis plus the cyclopropane fatty acyl phospholipid synthase that converts the UFA acyl chains of phospholipids to their cyclopropane derivatives. However, the source of the octadecenoic acid (C18:1), the UFA precursor of the C19 cyclopropane acid, was unclear. H. pylori genomes lack genes that could encode analogues of the known enzymes of anaerobic unsaturated fatty acid synthesis, FabA, FabQ and FabM. Also missing are the desaturase genes of oxygen-dependent UFA synthesis. However, H. pylori strain 26695 HP0773 encodes a possible homologue (31% amino acid identity) of the Neisseria UfaA protein which led us to test the possibility that HP0773 was involved in UFA synthesis. We report that expression of HP0773 (renamed FabX) allows a fabA E. coli UFA auxotroph to produce UFA in vivo. In vivo labeling with radioactive fatty acids of medium chain lengths plus reconstitution of the H. pylori UFA synthetic pathway in vitro showed that the branch point for UFA synthesis occurs at the decanoyl-ACP intermediate. That is, FabX takes a finished decanoyl-ACP and introduces a trans-2 double bond into this fully saturated intermediate. This reaction parallels that of acyl-CoA dehydrogenase, the first enzyme of the β-oxidation cycle of fatty acid degradation. FabX then isomerizes the trans-2 double bond to produce cis-3-decenoyl-ACP, the key intermediate in UFA synthesis. Thus, FabX reverses the normal fatty acid synthesis cycle at the C10 level to insert the cis double bond of the H. pylori UFAs.

RESULTS

N. gonorrhoeae ufaA was discovered as a gene that was highly expressed upon shift of aerobic cultures to anaerobic conditions (Isabella and Clark, 2011a). The gene was disrupted and the resulting mutant strain failed to grow under anaerobic conditions although aerobic growth was normal (Isabella and Clark, 2011). The encoded protein was annotated as a nitronate monooxygenase. Several such proteins have been shown to encode FabK enoyl-ACP reductases (Marrakchi et al., 2003; Massengo-Tiasse and Cronan, 2009; Zhu et al., 2013) which catalyze the NADPH-dependent reduction of the trans-2 double bond via an FMN cofactor in the last step of the fatty acid synthesis cycle rather than the annotated reaction. Moreover, function of UfaA under anaerobic conditions showed it could not be an oxygen-requiring enzyme. Alignment of UfaA with FabK proteins (Figure 1) led to testing rescue of growth by addition of fatty acids. This was successful, but only UFAs supported growth and thus the gene was named ufaA (Isabella and Clark, 2011b). No enzymology was done and thus it was unclear if the UfaA protein was solely responsible for UFA synthesis or required other N. gonorrhoeae proteins to perform this task.

Figure 1.

Figure 1

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Alignments of UfaA, FabX and CAC3580 (the putative Clostridium acetobutylicum FabX) and their comparison to two FabK enoyl-ACP reductases. Ca FabK is from C. acetobutylicum (CAC3576) whereas Ef FabK is EF2883 from Enterococcus faecalis. The overlined residues are those predicted to be involved in FMN binding. Expression of E. faecalis FabK has been shown to allow growth of an E. coli strain having a temperature-sensitive FabI enoyl-ACP reductase strain at the nonpermissive temperature (Zhu et al., 2013). Expression of the C. acetobutylicum CAC3576 protein also complemented growth of the E. coli FabI strain (Srinivas and Cronan, unpublished).

We began by studying the properties of UfaA and putative homologs from Clostridium acetobutylicum (CAC3580) and Helicobacter pylori (Figure 1). We were unable to detect activity in any of our preparations of the UfaA or C. acetobutylicum proteins. The reason for the lack of UfaA activity is unclear but the C. acetobutylicum lost its FMN cofactor during purification and it could not be replaced suggesting that the FMN binding site had somehow been lost. However, the H. pylori HP0773 protein (which we call FabX) had activity in E. coli and proved to be reasonably tractable in vitro. Hence we focused on this protein although sporadic loss of the FMN cofactor remains a problem.

FabX partially restores UFA synthesis to E. coli fabA mutant strains

The function of FabX was tested in parallel using two E. coli fabA mutant strains, CY57 and HW8 (Table S1) expressing FabX. No growth resulted without oleic acid supplementation (data not shown) as reported in the previous UfaA studies (Isabella and Clark, 2011). However, it remained possible that the recombinant plasmid allowed UFA synthesis, but that the levels of UFA synthesis were insufficient for growth as observed previously (Bi et al., 2013; Wang and Cronan, 2004). This possibility was tested by [1-14C]acetate labeling of the fatty acids synthesized by the fabA knockout strain HW8 expressing fabX. Synthesis of radioactive UFAs was readily detected by argentation thin layer chromatography whether or not a low dose of triclosan, a specific inhibitor of the E. coli FabI enoyl-ACP reductase, was present during labeling (Figure 2A). Note that FabX expression did not allow UFA synthesis by an E. coli fabB mutant strain (Figure S1).

Figure 2.

Figure 2

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In vivo Activity of FabX.

(A and B) Incorporation of [1-14C]acetate or medium chain length [1-14C]-labeled fatty acids into the membrane phospholipids of the wild-type E. coli strain YYC1273 or the E. coli fabA strain HW8 transformed with the empty pBAD24M vector or plasmid pBHK597 encoding H. pylori fabX. (A) Argentation TLC analysis of [1-14C]acetate-labeled E. coli fabA strain HW8 transformed with the H. pylori fabX. (B) Argentation TLC analysis of [1-14C]-labeled octanoate-, decanoate-, or dodecanoate-labeled E. coli fabA strain HW8 transformed with H. pylori fabX. All strains carried the AasS-encoding plasmid pBHK632 to allow the conversion of the exogenous fatty acids to the ACP thioesters required to enter the fatty acid synthetic pathway. Arabinose (ARA) was present at a concentration of 0.02% and IPTG at a concentration of 0.5 mM. Triclosan (TCL) was added at 0.1 mg/ml to partially inhibit the host FabI activity. Sat, SFA esters.

(C and D) Determination of double bond positions of the UFAs extracted from the wild-type YYC1273 and an E. coli fabA strain HW8 transformed with H. pylori fabX. The cultures were grown in RB medium to late-log phase at 37 °C. Growth of strain HW8/pBHK597 was supported by supplementation with the cyclopropane fatty acid, cis-9,10-methylenehexadecanic acid (17:0 cyc). The mass spectra are: panel C, methyl cis-9-hexadecenoic acid; panel D, methyl cis-11-octadecenoic acid. Each of the unsaturated esters gave a cleavage fragment of m/z (numbers in ovals) corresponding to the methyl end of the molecule and a second fragment corresponding to the ester end of the molecule (numbers in squares).

Although the expression of FabX in strain HW8 gave low levels of UFA (Table S2), sufficient material for identification of the double-bond positions and chain lengths of the UFAs by mass spectral analyses was obtained. The UFAs were cis-9-hexadecenoic and cis-11-octadecenoic acids (Figure 2C and 2D) in agreement with the argentation chromatography results (Figure 2A). These double bond positions indicated that, if the classical pathway was used, FabX would catalyze dehydration of 3-hydroxydecanoyl-ACP.

FabX catalyzes a novel pathway of UFA synthesis through reversal of the fatty acid synthetic cycle

To test if FabX utilized the classical mechanism we performed in vivo labeling studies analogous to those previously done in the Bloch laboratory (Goldfine and Bloch, 1961; Scheuerbrandt et al., 1961). These workers labeled Clostridium butyricum (now C. beijerincki) cultures with [1-14C-labeled octanoic acid or decanoic acid and analyzed the pattern of labeling of the cis-9-hexadecenoic and cis-11-octadecenoic acids synthesized (Scheuerbrandt et al., 1961). They reported that octanoate labeled the UFA species as well as the saturated fatty acids (SFAs) whereas labeled decanoic acid labeled mainly SFAs. Based on these results they proposed that the double bond moieties were introduced during the conversion of octanoyl chains to decanoyl chains followed by elongation by three or four acetate units to the cis-3-decanoyl intermediate (Scheuerbrandt et al., 1961). Note that although these papers led to the classical FabA/FabB pathway documented in E. coli, the data reported do not decisively establish the C. beijerincki pathway. This is because UFAs were formed from labeled dodecanoic and tetradecanoic acids, albeit at 3-fold lower levels than with octanoate (Goldfine and Bloch, 1961). A further complication is that the bacterium makes two sets of UFAs (Goldfine and Panos, 1971; Scheuerbrandt et al., 1961) and this was acknowledged in only one of the 1961 papers (although they appeared in the same issue). In summary the 1961 data could support mechanisms for C. beijerincki UFA synthesis other than the dehydration/elongation mechanism proposed (Scheuerbrandt et al., 1961) including that put forth in this paper. However, the subsequent work of Bloch and coworkers (Bloch, 1971) and others (Feng and Cronan, 2009; Guerra and Browse, 1990; Heath and Rock, 1996; Jiang et al., 2010; Nguyen et al., 2014) leaves no doubt that E. coli and numerous other bacteria use the classical pathway as originally proposed (Scheuerbrandt et al., 1961).

We performed similar radioactive labeling studies in E. coli, a bacterium that normally does not allow exogenous fatty acids to enter fatty acid synthetic pathway. However, expression of Vibrio harveyi acyl-ACP synthetase (AasS) allows exogenous fatty acids to enter the fatty acid synthetic pathway (Beld et al., 2014; Bi et al., 2013; Jiang et al., 2010). Hence, we transformed the E. coli fabA deletion mutant strain HW8 (which lacks the ability to convert UFA moieties to their cyclopropane derivatives) with plasmids encoding FabX and AasS and labeled the cells with [1-14C]-C8, -C10 or -C12 saturated fatty acids. As in the classical pathway [1-14C]octanoic acid was incorporated into both UFAs and SFAs. However contrary to the classical pathway [1-14C]decanoic acid was also incorporated into both UFAs and SFAs whereas [1-14C]dodecanoic acid labeled only SFAs (Figure 2B). These labeled UFA species migrated on argentation chromatography as esters of cis-9-hexadecenoic and cis-11-octadecenoic acids, the UFAs of E. coli. In contrast the E. coli strain that lacked FabX gave the expected conversion of 14C-octanoic acid to both UFA and SFA whereas 14C-decanoic acid and 14C-dodecanoic acid labeled only SFAs as expected (Bi et al., 2013; Jiang et al., 2010). The identities of the synthesized UFAs were confirmed by mass spectral analyses of the fatty acid methyl esters and their dimethyl disulfide adducts (Table S2). Since the double bond positions of the H. pylori UFAs were unknown (Geis et al., 1990) H. pylori strain 26695 was also analyzed (Figure S3). The double bonds of all three UFAs were located seven carbons from the methyl end. Hence, H. pylori synthesizes the same UFA species as the foreign host, E. coli.

The ability of FabX to catalyze incorporation of 14C-decanoic acid into UFA species having double bonds seven carbons from the methyl end indicated an unprecedented UFA synthetic pathway in which FabX had both acyl-ACP dehydrogenase activity and trans-2 to cis-3 isomerase activity. Decanoyl-ACP would undergo 2,3-dehydrogenation to trans-2-decenoyl-ACP followed by its isomerization to cis-3-decenoyl-ACP. This product would be elongated to cis-9-C16:1-ACP and cis-11-C18:1-ACP, the acyl chain species identified by mass spectral analysis of the H. pylori phospholipid-derived esters (Figure S2). The first of the FabX reactions has a parallel with that catalyzed by acyl-CoA dehydrogenase, the first enzyme of the β-oxidation pathway which produces trans-2-acyl-CoAs by 2,3-dehydrogenation of acyl-CoAs using an FAD cofactor. The isomerization reaction parallels those catalyzed by FabA, FabM and FabQ.

Enzymatic Properties of FabX

To test FabX for the postulated enzyme activities an N-terminal hexahistidine-tagged version of FabX was expressed in E. coli and was purified to homogeneity by affinity chromatography followed by size exclusion chromatography (Figure 3A & C). FabX is a monomeric flavoprotein of 40 kDa (Figure 3B). Tryptic peptide analysis gave 65% coverage of the peptides predicted from the DNA sequence (Figure 3D). The yellow cofactor was released by heat denaturation and identified as FMN (Figure S2).

Figure 3.

Figure 3

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Purification and structural characterization of FabX.

A. Gel exclusion chromatographic profile of the hexahistidine-tagged FabX run on a Superdex 200HR 10/30 column (GE Healthcare) eluted at 0.4 ml min−1. FabX was monitored at 280 nm and eluted at 15.26 min. OD280, optical density at 280 nm; mAu, milli-absorbance units.

B. Determination of FabX solution structure according to elution patterns of a series of standards (Bio-Rad). The standards were vitamin B12 (1.35 kDa), myoglobin (horse, 17 kDa), ovalbumin (chicken, 44 kDa), γ-globulin (bovine, 158 kDa) and thyroglobulin (bovine, 670 kDa). The elution position of FabX indicates an estimated molecular mass of 40 kDa based on graphic analysis of the standard curve. Kav denotes the partition coefficient.

C. SDS-PAGE analysis of purified FabX. The apparent molecular weight of His-tagged FabX was about 38 kDa. M: Molecular weight.

D. Mass spectrometric identification of FabX. The matching peptides are given in bold and underlined type.

FabX was assayed for the ability to convert decanoyl-ACP to unsaturated species by a gel electrophoretic mobility shift assay. The addition of FabX resulted in the production of a new acyl-ACP that migrated faster than the substrate decanoyl-ACP (Figure 4A). Moreover, as the concentration of FabX was increased a second product appeared which migrated much more slowly but faster than FabX (Figure 4B). The FabX plus decanoyl-ACP (22.5 μM) reaction products were separated and purified by ion exchange chromatography using a small cartridge (Figure 4D). The more rapidly migrating product eluted with 0.5 M LiCl, as expected for an ACP species and was identified as trans-2-decenoyl-ACP by its conversion to decanoyl-ACP upon addition of the E. coli FabI enoyl-ACP reductase and NADH (Figure 4B & 4C). The slowly migrating product appeared in the flow-through of the ion exchange cartridge. Samples were digested with trypsin and the resulting peptides analyzed were from both FabX and ACP. The bound acyl-ACP was thought to be cis-3-decenoyl-ACP because only this acyl-ACP species can enter the unsaturated chain elongation cycle. This product was identified using two assays. The flow-through of the ion exchange cartridge was subjected to sodium methoxide catalyzed transesterification to give the methyl ester which was then converted to the dimethyl disulfide adduct for double bond position determination. As expected the bound fatty acid was 3-decenoic acid (Figure 4E). Purified E. coli fatty acid synthesis proteins were then used to test elongation of the flow-through product by E. coli FabB. FabD was incubated with [2-14C]malonyl-CoA and holo-ACP to generate [2-14C]malonyl-ACP (Figure 4F, lane 1). Addition of FabB plus FabX to [2-14C]malonyl-ACP failed to result in conversion of the label to a [14C]-labeled acyl-ACP (Figure 4F, lane 3), indicating that purified FabX did not carry a bound acyl-ACP that could serve as an elongation substrate. Addition of FabB plus the in vitro reaction product formed with FabX (0.83 μM) resulted in formation of [14C]-labeled 3-oxododecanoyl-ACP by FabB-catalyzed elongation of decanoyl-ACP with [2-14C]malonyl-ACP (Figure 4F, lanes 2 and 4). However, when FabX at high concentration (22.5 μM) in the complex with cis-3-decenoyl-ACP was incubated with FabB and [2-14C]malonyl-ACP, the slowly migrating band became radioactive. Since incorporation of label from malonyl-ACP indicated that elongation had occurred, it seemed likely that this labeled product was a complex that contained FabX and a bound [14C]-labeled 3-oxo-cis-5-C12:1-ACP. Note that FabB is unable to elongate trans-2-decenoyl-ACP (Heath and Rock, 1996). Thus, it seemed that in contrast to FabA (Heath and Rock, 1996) FabX-catalyzed production of cis-3-C10:1-ACP did not depend on the presence of FabB. When the reaction contained a high concentration of FabX (22.5 μM), no [14C]-labeled 3-oxododecanoyl-ACP was formed indicating that FabX had converted all of the decanoyl-ACP to the unsaturated species trapped in the complex (Figure 4F, lane 5). A FabX preparation that had lost its FMN cofactor during dialysis was inactive. Attempts to scale up a nonradioactive version of the Figure 3F, lane 5 reaction to allow mass spectral analyses were unsuccessful, probably due to decarboxylation of the unstable 3-oxo acid during extraction and derivation. Like FabA (Heath and Rock, 1996), FabX does not show strict chain length specificity in vitro in that it catalyzed dehydrogenation of octanoyl-ACP and dodecanoyl-ACP (Figure S4).

Figure 4.

Figure 4

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Identification of the FabX reaction products formed in vitro.

(A) The catalytic properties of FabX were tested in vitro with decanoyl-ACP as the substrate. The reaction mixtures were as described in Experimental Procedures. After incubation at 37°C for 20 min the reaction products were resolved by conformationally sensitive gel electrophoresis on 18% polyacrylamide gels containing 2.5 M urea. The minus sign denotes a reaction lacking FabX (lane 1). The horizontal triangle over the right-hand represents FabX concentrations in a series (0.83, 2.5, 7.5 and 22.5 μM). Lane 1 lacked FabX.

(B and C) Demonstration of the FabX dehydrogenase activity. (B) Lane 1 is the substrate decanoyl-ACP (100 μM, 20 μl); Lane 2 is the in vitro reaction product of decanoyl-ACP with FabX (22.5 μM, 20 μl) (the same reaction as Lane 5 of panel A) and lane 3 is FabX (22.5 μM, 20 μl) in the absence of substrate. Lane 4 is the product eluted from the reaction of Lane 2 with 0.5 M LiCl (Lane 6 of panel D is the same sample) whereas Lane 5 denotes the product formed after the addition of E. coli FabI and NADH to a sample of the product of Lane 4. (C) NADH oxidation activity of E. coli FabI on different substrates. The E. coli FabI reaction was monitored at 340 nm (NADH oxidation) using authentic trans-2-decenoyl-ACP (triangles) or trans-2-decenoyl-ACP (squares) purified from an in vitro FabX reaction (Lane 4 of panel B). The diamond symbols denote the background observed in the absence of substrates. The curves in the figure were adjusted to the same zero time absorbance. The data are the means of three independent assays.

(D–F) Detection of FabX isomerase activity. (D) The mixture of products formed in vitro by reaction of FabX (22.5 μM) with decanoyl-ACP (Lane 1, the same sample as Lane 5 of panel A) was analyzed by ion exchange chromatography using Vivapure D Mini H columns eluted in steps with the indicated LiCl concentrations. (E) Mass spectroscopy of the cleavage products of the dimethyl disulfide adducts of fatty acid methyl esters extracted from the flow-through mixture (lane 2 of panel D) and the cis-3-decenoic acid standard. The unsaturated ester gave a cleavage fragment of m/z 145 (oval number) corresponding to the methyl end of the molecule plus a second fragment m/z 133 (square number) corresponding to the ester end of the molecule. The spectral data were identical with those obtained using a commercial sample of cis-3-decenoic acid. (F) Reconstruction of fatty acid synthesis in vitro indicating cis-3-decenoyl-ACP production. 14C-Labeled malonyl-ACP was first synthesized in all reactions as described in Experimental Procedures. The other indicated proteins or reaction mixtures were subsequently added to the 14C-labeled reaction mixtures of lanes 2–5 followed by incubation at 37°C for an additional 20 min. Note that in order to allow FabX to react with decanoyl-ACP before elongation by FabB, the reaction mixtures containing C10-ACP and FabX in Lanes 4 and 5 were incubated at 37°C for 20 min before addition to the 14C-labeled reaction mixtures. The reaction products were analyzed as in panel A. The 3-oxo-C12-ACP label denotes 3-oxododecanoyl-ACP.

Note that E. coli was the source of the ACP used in most experiments because methods for separating its acylated species are well developed (Cronan and Thomas, 2009). Moreover, the E. coli and H. pylori seem largely interchangeable because H. pylori ACP has been shown to functionally replace E. coli ACP in vivo (De Lay and Cronan, 2007). The decanoyl-thioesters of H. pylori and E. coli ACPs in the FabX dehydrogenase reaction and found the two substrates to have similar activities. For unknown reasons the H. pylori ACP decanoyl and trans-2-decenoyl thioesters failed to resolve in the gel system that resolves the two E. coli acyl-ACPs (Figs. 4 and 5) and thus FabX-catalyzed dehydrogenation of H. pylori decanoyl-ACP was determined by running timed reactions in parallel with E. coli decanoyl-ACP followed by assay of the trans-2-decenoyl-ACP produced using E. coli FabI as in Fig. 4C. H. pylori decanoyl-ACP was modestly more active (about two-fold) in the FabX reaction than E. coli decanoyl-ACP.

Figure 5.

Figure 5

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Oxygen is the sole electron acceptor able to support the FabX dehydrogenase reaction in vitro.

A. Two identical reaction mixtures lacking FabX were degassed and left in the anaerobic chamber for 48 h to ensure anaerobiosis. FabX was added and allowed to react for 30 min at 37°C following which oxygen was bubbled through one of the tubes for 30 min. The reactions were then placed on dry ice, removed from the chamber and subjected to 18% polyacrylamide gel electrophoresis containing 2.5 M urea. Decanoyl-ACP and trans-2-decenoyl-ACP standards were loaded in the first and last lanes, respectively.

B. A gradient of oxygen concentrations was generated by mixing anaerobic and air-saturated (21% oxygen) reaction mixtures in different proportions in the anaerobic chamber. FabX was then added. The reactions allowed to proceed for 30 min at 37°C and the acyl-ACPs were the precipitated with trichloroacetic acid. The precipitates were recovered and analyzed as in panel A. Decanoyl-ACP and trans-2-decenoyl-ACP standards were loaded in the first and last lanes, respectively.

C. Ability of various alternative electron acceptors to support the FabX dehydrogenase reaction. The compounds tested were in great excess over FabX and decanoyl-ACP. They were present at 1 mM except cytochrome c (1 mg/ml). The reaction mixtures containing these compounds were made anaerobic as in panel A. The reactions were initiated by FabX addition and were incubated at 37 °C for 30 min in the chamber before being placed on dry ice until analysis by polyacrylamide gel electrophoresis as in panel A. Decanoyl-ACP and trans-2-decenoyl-ACP standards were loaded in the first and last lanes, respectively.

Oxygen acts as the in vitro electron acceptor in FabX catalysis

The FabX dehydrogenation reaction is strongly analogous to that of the highly studied acyl-CoA dehydrogenases of fatty acid β-oxidation although the proteins have different flavin cofactors and essentially no sequence similarity. Dehydrogenation of a C-C bond is a challenging oxidation reaction that requires the rupture of two kinetically stable C-H bonds. This requires a more positive reduction potential than those of NAD+ or NADP+. The FAD cofactor of the well-studied mitochondrial medium chain length acyl-CoA dehydrogenase has a reduction potential (E0′) of −208 mV and substitution of the FAD with analogues having more negative redox potentials (e.g., -280 mV for deaza analogues) result in inactive enzymes (Thorpe and Massey, 1983). Hence we expect that the FMN cofactor in FabX must have a reduction potential of -208 mV or greater. FabX is catalytic in our assays. For example in 0.83 μM FabX converted >50 μM decanoyl-ACP to trans-2-decenoyl-ACP (Figure 4A) and thus the FMNH2 cofactor must have been reoxidized. The only oxidant present in our reactions was oxygen which has long been known to directly oxidize reduced flavins with the formation of hydrogen peroxide (Fitzpatrick, 2004; Massey, 2000).

Oxygen was demonstrated to be the oxidant by experiments performed in an anaerobic chamber (Figure 5A). After the reaction mixtures had been made anoxic by incubation in the chamber for two days, addition of FabX gave only a trace of activity that was readily attributed to oxygen present in the enzyme solution. However, upon bubbling the reaction with air much of the decanoyl-ACP substrate was converted to trans-2-decenoyl-ACP (Figure 5A).

Since H. pylori is a microaerophilic bacterium we tested if oxygen at the low concentrations (3 to 7% O2) that allow growth of this bacterium would suffice for the FabX dehydrogenation reaction (Figure 5B). Good activity was observed at 3% O2, although higher oxygen concentrations gave increased activity (Figure 5B). Finally we tested eight possible electron acceptors including the quinone/menaquinone analogue, plumbagin, under anaerobic conditions and found that none supported the FabX dehydrogenation reaction (Figure 5C).

We attempted to use a sensitive fluorescent hydrogen peroxide assay as a more facile means to directly measure FabX dehydrogenase activity. However, attempts to detect hydrogen peroxide formation dependent on the presence of FabX and decanoyl-ACP were only partially successful due to interference by components of the reaction mixtures. For example, spiking reaction mixtures with a known concentration of hydrogen peroxide resulted in only about 10% recovery indicating quenching of peroxide (and/or the assay) by reaction components. In other cases the rate of peroxide formation decreased as the dehydrogenation reaction proceeded (as assayed by gel electrophoresis) suggesting quenching by a reaction product. Indeed, we generally recovered only 5–10% of the peroxide expected from the amount of decanoyl-ACP converted to trans-2-decenoyl-ACP and therefore abandoned this approach.

DISCUSSION

Our in vivo experiments demonstrate that H. pylori FabX can functionally replace E. coli FabA in synthesis of UFAs indicating that FabX isomerizes trans-2-decenoyl-ACP to cis-3-decenoyl-ACP. These results suggested that like FabA (Bloch, 1971), FabX catalyzes both the synthesis of trans-2-decenoyl-ACP and its isomerization. However, the radioactive fatty acid labeling studies demonstrated that FabX did not use the classical pathway to make trans-2-decenoyl-ACP. Analysis of the C16 and C18 UFAs of both E. coli expressing FabX and H. pylori indicated that the cis double bond was inserted between carbons 3 and 4 of a C10 acid. The only manner in which this could occur was for FabX to catalyze a 2,3-dehydrogenation analogous to the acyl-CoA dehydrogenase reaction (Ghisla and Thorpe, 2004; Kim and Miura, 2004) to give trans-2-decenoyl-ACP followed by isomerization to cis-3-decenoyl-ACP (Figure 6).

Figure 6.

Figure 6

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Proposed Pathway for FabX-dependent UFA formation in H. pylori.

In H. pylori, UFA synthesis proceeds from a pathway branch at the decanoyl-ACP stage. FabX dehydrogenates decanoyl-ACP to trans-2-decenoyl-ACP plus FMNH2 and isomerizes the trans-2-decenoyl-ACP intermediate to cis-3-decenoyl-ACP. FabF is required for the elongation of cis-3-decenoyl-ACP to allow this intermediate entry into the fatty acid synthesis cycle to produce the long-chain UFAs of the phospholipids.

Oxygen was the oxidant required for FabX catalysis in vitro (Figure 5). The mammalian acyl-CoA dehydrogenases use bulky residues (usually tryptophan) to shield their FAD moieties from oxygen (Ghisla and Thorpe, 2004; Kim and Miura, 2004). However shielding would not be required for the putative C. acetobutylicum FabX since the clostridia are obligate anaerobes and for N. gonorrhoeae UfaA which functions only under anaerobic conditions (Isabella and Clark, 2011a, b). It should be noted that FabX and similar proteins such as FabK are often annotated as nitronate monooxygenases (generally under the prior name, 2-nitropropane dioxygenase). These proteins are placed in the same protein superfamily as acyl-CoA dehydrogenases and acyl-CoA oxidases and share a common domain structure (Ghisla and Thorpe, 2004; Kim and Miura, 2004). Acyl-CoA oxidases use oxygen as a substrate and thus the finding that oxygen can access the FabX flavin is not unprecedented. The hydrogen peroxide produced is toxic due to its reactivity, but H. pylori can cope using catalase and DNA repair capabilities (O’Rourke et al., 2003; Olczak et al., 2002). To avoid peroxide formation an unknown protein could shuttles electron from FabX to the membrane quinone pool as in the mitochondrial acyl-CoA dehydrogenases. The mitochondrial dehydrogenases transfer electrons from their bound FADH2 to the FAD of a soluble electron-transfer flavoprotein which relays the electrons to a third FAD enzyme, the membrane-bound electron transfer flavoprotein:ubiquinone oxidoreductase (Watmough and Frerman, 2010). Thus the electrons extracted from the acyl chains end up in the ubiquinone pool of the main respiratory chain. Another possibility is that a small molecule such as fumarate acts as an electron shuttle from FabX to a membrane-bound fumarate reductase and hence to menaquinone (Cecchini et al., 2002; Lancaster and Simon, 2002). However, none of the possible acceptors we tested allowed FabX activity under anaerobic conditions (Figure 5C). Although the microaerophilic conditions required for growth of H. pylori are sufficient for FabX activity in vitro, obligate anaerobes such as the clostridia would need an alternative means to reoxidize the FabX flavin. A good possibility is flavin-based electron bifurcation performed by cytoplasmic multienzyme complexes from anaerobic bacteria and archaea (Buckel and Thauer, 2013; Chowdhury et al., 2014; Nitschke and Russell, 2012). Indeed, one of these systems has been reported to be reversible (Chowdhury et al., 2014).

An unanswered question is why cis-3-decenoyl-ACP is observed only in a complex with FabX whereas the trans-2-decenoyl-ACP intermediate is released from the enzyme. The FabX complex with cis-3-decenoyl-ACP was first detected by tryptic peptides derived from both FabX and ACP. The complex is quite stable in that it survives electrophoresis into 2.5 M urea and exposure to an ion exchange resin that binds free ACP species. However the two proteins are resolved by SDS-gel electrophoresis indicating a non-covalent complex. The complex is not a simple aggregate because E. coli FabB can elongate the cis-3-decenoyl-ACP with [2-14C]malonyl-ACP. One possibility is that the putative H. pylori FabF elongation enzyme (HP0558) interacts with the complex to elongate and release cis-3-decenoyl-ACP whereas E. coli FabB would lack releasing ability. However, other possibilities exist such as the small proteins reported to interact with E. coli FabA (Rock et al., 1996; Sugai et al., 2001).

FabX seems very likely to be essential for growth of H. pylori. Salama and coworkers (Baldwin et al., 2007; Salama et al., 2004) have mapped over 5300 transposon insertions into H. pylori genomes without finding an insertion into HP0773 (fabX) and the gene is strictly conserved in the genomes of H. pylori strains isolated from extremely diverse origins (data for over 175 strains were collated) (Baldwin et al., 2007; Didelot et al., 2013; Gressmann et al., 2005; Kawai et al., 2011; McClain et al., 2009). Extrapolation of the numbers of core genes among these strains give values ranging from 786 to about 1100 illustrating the unusual plasticity of H. pylori genomes and the high rate of gene flux in and out of these genomes. Given the remarkably high rates of mutation and recombination in H. pylori, it seems likely that if HP0773 (fabX) is not essential for growth, at least some of the H. pylori strains should have lost the gene. The fabX gene is found in Helicobacter species other than H. pylori. However, caveats to this conclusion are the genome plasticity, the lack of clustering of pathway genes and the sequence relatedness of FabX to the FabK enoyl-ACP reductases (Figure 1). Note that Helicobacter species contain highly probable fabI genes (e.g., HP0195 in H. pylori).

FabX homologs (e.g. UfaA) are reportedly found across the bacterial kingdom mostly, if not entirely, in organisms that are facultative or obligate anaerobes (Isabella and Clark, 2011b). None of the putative FabX-containing bacteria contain a functional FabA or FabM homologue (Isabella and Clark, 2011b) and FabX may explain how these bacteria synthesize UFAs. Indeed, Isabella and Clark (Isabella and Clark, 2011b) argue that N. gonorrhoeae inactivated its fabA and fabB genes in favor of UfaA. Note that FabX-dependent synthesis of UFAs seems likely in many pathogenic bacteria in addition to H. pylori, pathogenic Clostridium spp., Campylobacter spp. and Burkholderia spp. Therefore, new antibacterial agents could be targeted at this key UFA synthesis enzyme.

EXPERIMENTAL PROCEDURES

Radioactive Labeling, phospholipid extraction and fatty acid analysis

The cultures were grown at 37 °C in RB medium (1% tryptone, 0.1% yeast extract, 0.5% NaCl) to late log-phase, the phospholipids were extracted by the method of Bligh and Dyer (Bligh and Dyer, 1959), and the fatty acid compositions were analyzed by mass spectroscopy. The position of the double bond in the UFA was determined by GC-MS analysis of dimethyl disulfide adducts prepared from fatty acid methyl ester samples. GC-MS analysis was performed at the Carver Metabolomics Center of the University of Illinois by GC-MS as described previously (Bi et al., 2013; Feng and Cronan, 2009).

For analysis of radioactive fatty acids, 0.1 ml of a culture grown overnight in LB medium was transferred into 5 ml of RB medium containing 0.01% of the cyclopropane fatty acid, cis-9,10-methylenehexadecanic acid and IPTG (0.5 mM). The cultures were grown for 1 h, 5 μCi of sodium [1-14C]acetate was added and growth was allowed to continue for 5 h. To partially inhibit E. coli FabI activity, triclosan was added at 0.1 μg/ml to strain HW8 expressing FabX. [1-14C]octanoic acid, [1-14C]decanoic acid and [1-14C]dodecanoic acid (each at 50–55 mCi/mmol) were similarly used to label the phospholipids of derivatives of strain YYC1273 and HW8/pBHK597 that expressed the AasS acyl-ACP synthase. The strains were grown in RB medium containing 15 μM labeled fatty acid, ampicillin, gentamicin and arabinose (0.02%). The cellular phospholipids were then extracted and the acyl chains converted to their methyl esters which were separated by argentation thin layer chromatography and detected by autoradiography as described previously (Bi et al., 2013; Bi et al., 2014). Radiolabeled fatty acids were purchased from Moravek Biochemicals, Inc. All thin layer plates were scribed into lanes to prevent cross-contamination.

Assay of FabX activity in vitro

FabX activity was assayed in the reactions containing 100 mM sodium phosphate (pH 7.2), 100 μM decanoyl-ACP and different concentrations of FabX in a volume of 20 μl. The reactions were initiated by adding FabX and incubated at 37 °C for 20 min. The reactions were immediately quenched by addition of an equal volume of 10 M urea and stored on dry ice until analysis. The reaction samples were mixed with gel loading buffer and analyzed by conformationally sensitive gel electrophoresis on 18% polyacrylamide gels containing a urea concentration optimized for the separation (Post-Beittenmiller et al., 1991). Decanoyl-ACP was synthesized using Vibro harveyi acyl-ACP synthetase AasS as described previously (Bi et al., 2013; Cronan and Thomas, 2009).

The reaction products formed in vitro with FabX (22.5 μM) were separated and purified using Vivapure D Mini H spin columns (Sartorius) according to the manufacturer’s recommendations. Briefly, the spin columns were equilibrated first with equilibration buffer (100 mM sodium phosphate, pH 7.2). The reaction mixtures were loaded and discontinuously eluted with a 100 mM sodium phosphate (pH 7.2) buffer containing 100, 200, 300 or 500 mM NaCl. The flow-through and each eluted fraction were desalted by overnight dialysis. The trans-2-decenoyl-ACP eluted at 500 mM NaCl was detected by the addition of enoyl-ACP reductase E. coli FabI, which catalyzes the reduction of the double bond to produce the saturated decanoyl-ACP. The NADH-dependent FabI activity was detected by NADH oxidation as described previously (Massengo-Tiasse and Cronan, 2008). Briefly, the FabI enoyl-ACP reductase activity was monitored spectrophotometrically by decrease in absorbance at 340 nm. Each 100 μl reaction was performed in disposable UV-transparent microcuvettes obtained from Brand-Tech Scientific. The activity assays contained 200 μM NADH, 100 ng of the purified E. coli FabI, different substrates (100 μM trans-2-decenoyl-ACP standard or the reaction product containing trans-2-decenoyl-ACP eluted at 500 mM NaCl) and 0.1 M LiCl in a 0.1 M sodium phosphate buffer (pH 7.0).

The production of cis-3-decenoyl-ACP in the complex was detected by mass spectroscopic analysis of the position of the double bond of the fatty acyl chains, extracted from the flow-through mixtures containing the complex. The mixtures were dried under nitrogen and the acyl chains of in vitro products were first converted to their methyl esters by sodium methoxide in methanol and then to their dimethyl disulfide adducts followed by analysis of gas chromatography-mass spectroscopy as described previously (Bi et al., 2012; Bi et al., 2013; Feng and Cronan, 2009). The identification of cis-3-decenoyl-ACP was also confirmed by its reaction with FabB, which catalyzes the elongation of cis-3-decenoyl-ACP (Feng and Cronan, 2009) but not of trans-2-decenoyl-ACP (Heath and Rock, 1996).

Fatty acid synthesis in vitro

FabX activity was characterized using purified E. coli enzymes to catalyze specific steps in fatty acid biosynthesis. To produce 14C-labeled malonyl-ACP, the fatty acid synthesis assay mixtures, which contained 0.1 M sodium phosphate (pH 7.0), 2 mM β-mercaptoethanol, 100 μM holo-ACP and 45 μM [2-14C]malonyl-CoA (specific activity, 55 mCi mmol−1) and E. coli FabD (0.5 μg) in a final volume of 20 μl, were incubated at 37°C for 15 min. Then purified His-tagged E. coli FabB (0.3 μg/assay) plus decanoyl-ACP (100 μM) or FabB (0.3 μg) plus FabX (22.5 μM) were added to the previous 14C-labeled reaction mixtures for control experiments. The reaction mixtures (10 μl reaction volume containing 100 μM C10-ACP and FabX), were first incubated at 37°C for 20 min before FabB (0.3 μg/assay) was also added to in order to allow FabX to react with C10-ACP before it could be elongated by FabB to 3-oxododecanoyl-ACP. After an additional incubation at 37°C for 20 min, the reactions were stopped by placing the tubes in an ice slush. Samples of the mixtures were mixed with gel loading buffer and analyzed by conformationally sensitive gel electrophoresis on 18% polyacrylamide gels containing a urea concentration optimized for the separation (Post-Beittenmiller et al., 1991). The gels were fixed, soaked in Enlightning (DuPont) dried and exposed to X-ray film.

Supplementary Material

1
2

SIGNIFICANCE.

Synthesis of unsaturated fatty acids is essential for most bacterial species. We have demonstrated a new pathway of unsaturated fatty acid synthesis that is catalyzed by an enzyme, FabX, that has dual dehydrogenase/isomerase activities. This unprecedented combination of enzyme activities reverses the usual fatty acid synthesis cycle at the C10 stage. The properties of FabX can explain how many bacterial species lacking the canonical pathway enzymes are able to produce the unsaturated fatty acid-containing phospholipids required for functional cell membranes. Given the prevalence of putative fabX genes in pathogenic bacteria, the enzyme could be a worthwhile target for development of antibacterial compounds.

Highlights.

  • (UFA) biosynthesis is required to maintain membrane function in bacteria

  • Many bacteria lack the canonical unsaturated fatty acid UFA synthetic pathway

  • An enzyme that inserts a double bond into a fully saturated acyl chain is reported

  • The enzyme has both dehydrogenase and isomerase activities

Acknowledgments

The authors have no Conflicts of Interest to report. This work was supported by a grant from National Natural Science Foundation of China (31570053 to H. B.), the start-up package from Nanjing Medical University (to H. B.) and National Institutes of Health Grant AI15650 from National Institute of Allergy and Infectious Diseases (to J. E. C.). We thank Dr. V. Isabella and Prof. V. Clark for communicating their findings before publication. We thank Prof. James Imlay for his advice on redox chemistry and use of his anaerobic chamber. We thank Drs. Peter Yau, Alexander Ulanov and Lucas Li of the Carver Biotechnology Center, University of Illinois, for help with mass spectrometric analyses.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, four figures, and two tables and can be found with this article online at

AUTHOR CONTRIBUTIONS

H.B., L. Zh. and J.C. designed research; H.B., L. Zh, J.J. and L. Ze. performed research; H.B., L. Zh. and J.C. analyzed data and wrote the paper. Both first authors designed experiments and performed key experiments (H.B. in the early work and L. Zh. In the later work).

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Supplementary Materials

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NIHMS830960-supplement-1.pdf (506.8KB, pdf)
2
NIHMS830960-supplement-2.pdf (2.6MB, pdf)

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