Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Structure. Author manuscript; available in PMC 2013 May 9.
Published in final edited form as:
PMCID: PMC3376755

Staphylococcus aureus FabI: Inhibition, Substrate Recognition and Potential Implications for In Vivo Essentiality


Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a serious health threat worldwide, and novel antibiotics are therefore urgently needed. The enoyl-ACP reductase (saFabI) is essential for the S. aureus fatty acid biosynthesis and, hence, serves as an attractive drug target. We have obtained a series of snapshots of this enzyme which provide a mechanistic picture of ligand and inhibitor binding including a dimer-tetramer transition combined with extensive conformational changes. Significantly, our results reveal key differences in ligand binding and recognition compared to orthologous proteins. The remarkable observed protein flexibility rationalizes our finding that saFabI is capable of efficiently reducing branched-chain fatty acid precursors. Importantly, branched-chain fatty acids represent a major fraction of the S. aureus cell membrane and are crucial for its in vivo fitness. Our discovery thus addresses a long-standing controversy regarding the essentiality of the fatty acid biosynthesis pathway in S. aureus and validates saFabI as a drug target.


Resistant strains of the Gram-positive human pathogen Staphylococcus aureus emerged only two years after the introduction of the antibiotic methicillin in 1959. Since then, methicillin-resistant S. aureus (MRSA) infections have become an increasing problem in hospitals worldwide. To treat severe MRSA infections, vancomycin is currently used as the drug of last resort (Pantosti and Venditti, 2009). Thus, the initial occurrence of a vancomycin-resistant S. aureus (VRSA) strain in June 2002 is especially alarming (Sievert et al., 2008). Unfortunately, antibiotic resistance has drastically outpaced new antibiotic discovery, which flourished between the 1940s and 1960s. Between 1962 and 2000 only a few novel scaffolds like the carbapenems were approved, and novel antibiotics with alternative modes of actions are therefore urgently needed. Accordingly, from 2000 onwards oxazolidinones (e.g. linezolid), the lipopeptide daptomycin and the mutilins were introduced (Fischbach and Walsh, 2009).

One strategy that targets the bacterial cell envelope involves the inhibition of the type II fatty acid biosynthesis pathway (FAS II), which is responsible for the de novo production of phospholipid precursor molecules (Payne et al., 2002). In contrast to the large multifunctional mammalian FAS I synthase, the FAS II system of bacteria, protozoa and plants consists of individual monofunctional enzymes, allowing selective inhibition (Payne et al., 2001). In this pathway, the fatty acid chain, which is attached to the acyl carrier protein (ACP), is elongated by two carbon atoms per cycle (Figure 1). The final reduction is catalyzed by the NAD(P)H-dependent trans-2-enoyl-ACP reductase (FabI), which is known to play a key regulatory role in this pathway (Heath and Rock, 1995; Xu et al., 2008). This enzyme is highly conserved across many pathogens, however, the S. aureus enoyl-ACP reductase (saFabI) is the only known FabI with a clear preference for NADPH (Heath et al., 2000; Priyadarshi et al., 2010; White et al., 2005; Xu et al., 2008). The clinical success of FabI inhibitors, such as the first-line tuberculosis prodrug isoniazid (Lu and Tonge, 2008) and several compounds currently in phase I clinical trials (Gerusz, 2010), validates this enzyme as an attractive drug target. Furthermore, the FabI diphenyl ether inhibitor triclosan (TCL) is recommended as topical antiseptic to reduce MRSA skin colonization (Bamber and Neal, 1999).

Figure 1
The bacterial type II fatty acid biosynthesis pathway as a drug target

Recently, however, Brinster et al. challenged the validity of FAS II inhibitors as Streptococcus agalactiae can utilize exogenous fatty acids from the host blood serum to survive inhibition of its FAS II machinery (Brinster et al., 2009). Whether this resistance mechanism extends to all Gram-positive bacteria including S. aureus is currently controversially discussed (Balemans et al., 2010; Parsons et al., 2011). Importantly, S. aureus remains sensitive to FabI inhibitors in vivo, as shown in several rodent models (Balemans et al., 2010; Escaich et al., 2011; Park et al., 2007; Payne et al., 2002).

To investigate the suitability of saFabI as a drug target, we have structurally and biochemically characterized this enzyme with respect to inhibitor binding, conformational flexibility, ligand binding mechanism, cofactor and substrate specificity, quaternary structure and cooperativity. We identified significant differences in the S. aureus and Bacillus FabIs compared to the classical FabI proteins from organisms such as Escherichia coli, Burkholderia pseudomallei and Francisella tularensis; and of our structures with respect to the recently determined S. aureus FabI structures in complex with triclosan and in its apostate (Priyadarshi et al., 2010). Importantly, our analysis revealed that the substrate specificity of saFabI displays an increased specificity for branched-chain (BCFA) relative to straight-chain fatty acid (SCFA) precursors (Figure S1), consistent with the high content of BCFAs in the membranes of Staphylococci and Bacilli (Kaneda, 1991). Since BCFAs, which are barely found in human serum (Holman et al., 1995), are likely required for S. aureus fitness in vivo, we conclude that saFabI is a suitable target for drug discovery.


Overall structure

To provide a molecular basis for the rational design of compounds that target saFabI, we solved the structures of ternary FabI complexes with the cofactor NADP+ and the diphenyl ether inhibitors triclosan (TCL), 5-chloro-2-phenoxyphenol (CPP) or 5-ethyl-2-phenoxyphenol (EPP), respectively (Figures 1, S1 and S2). As a member of the short-chain dehydrogenase/reductase (SDR) superfamily, saFabI contains an extended Rossmann-fold comprised of a central seven-stranded parallel β-sheet surrounded by three α-helices on either side (Figure 2B) (Grimm et al., 2000; Lu and Tonge, 2008). Furthermore, the structures of our saFabI inhibitor complexes clearly show the presence of homo-tetramers (Figure 2A) as observed in other organisms (White et al., 2005). The closest relationship is found with the FabI structures from the Gram-positive organisms Bacillus anthracis and B. subtilis as well as with the incorrectly designated “FabL” from B. cereus which is 100% identical to B. anthracis FabI (Figure 3).

Figure 2
Overall structure and flexibility of saFabI
Figure 3
Secondary structure of saFabI and alignment with similar proteins

In addition, we solved two apo saFabI structures which display unexpected flexibility in close proximity to the binding site (Figure 2C). In one of the two apo structures all four monomers (apo-2; nomenclature of all structures according to Tables S1 and S2) differ in their active site (pairwise rmsd of 0.84 Å) and display conformations that, to our knowledge, have never been observed before. Compared to the inhibitor-bound complexes, three regions - the active site and two substrate-binding loops (Figure 3) - are either disordered or visible in a rearranged architecture. In contrast, previously published apo structures of FabI proteins from Gram-negative bacteria (PDB codes: 2JJY, 2P91, 2WYU, 3EK2, 3GRK, 3K2E) show disorder in only one region, the substrate-binding loop.

SaFabI inhibition

In contrast to the previously described saFabI-NADP+-TCL structure (PDB code: 3GR6) (Priyadarshi et al., 2010), our high-resolution saFabI complex structures with the unsymmetrically substituted inhibitors EPP and CPP unequivocally reveal a similar binding mode of diphenyl ethers including triclosan to saFabI as reported for homologous FabI proteins (Lu and Tonge, 2008; Xu et al., 2008) which allows the formation of two key hydrogen bonds connecting the A-ring hydroxyl group to Tyr157 and to the 2′-OH of the nicotinamide ribose at 2.5 ± 0.1 and 2.6 ± 0.1 Å, respectively (Figure 4B). TCL, CPP and EPP bind to a pocket composed of NADP+ and several mainly hydrophobic residues with average distances of 3.8 ± 0.4 Å (Figures 4B and C). Notably, the carbonyl oxygen of Ala97 forms a geometrically favorable halogen bond (Bissantz et al., 2010) with the TCL B-ring chlorine at position 4 (3.2 ± 0.1 Å). At the same time the chlorine is weakly hydrogen bonded to the amide NH group of Ala97 (3.2 ± 0.1 Å, Figure 4B). Accordingly, TCL exhibits an almost 3-fold lower Ki*,app value as compared to CPP, which lacks the two B-ring chlorines and is rotated by 11 ± 2° into the binding crevice (Table 1, Figure 4D and Supplemental Results).

Figure 4
SaFabI inhibition with diphenyl ethers
Table 1
Potency of saFabI inhibition by diphenyl ether analogues.

Induced fit ligand binding

SaFabI undergoes several major conformational changes upon cofactor and inhibitor binding, which have, to our knowledge, never been observed previously for its Gram-negative FabI orthologs, thus allowing us to describe a fundamentally expanded mechanism for ligand binding. All residues interacting with the diphenyl ether inhibitors are located in three distinct regions (Figure 3), which exhibit substantial flexibility prior to cofactor and inhibitor binding. As described for numerous FabI orthologs and other SDR proteins, the substrate-binding loop (SBL, 194–204) is disordered or rearranged in our apo structures (Grimm et al., 2000; Tipparaju et al., 2008). In contrast to other FabI structures, however, the saFabI structures reveal flexibility of a second substrate-binding loop (SBL-2, 94–108) as well as the active site loop (ASL, 147–157).

Superposition of the apo-1 structure with TCL-2 shows that the active site is wide open prior to cofactor and inhibitor or substrate binding (Figure 5A). In addition to the three aforementioned loop regions, the C-terminal SBL extension α7 is disordered. The apo-2 structure provides informative snapshots of α7’s motion on its way to the ternary complex conformation as every subunit shows a different arrangement of this helix (Figure 5B-F and Movie S1). In one subunit, this helix is shifted ~13 Å away from the active site (Figure 5B). When helix α7 moves towards the protein core, it is still rotated about 105° relative to the ternary complex conformation (Figure 5C) and assumes its final conformation via rotation angles of 70° and 20° (Figure 5D–F). Intermittently, the adjacent helix α8 is elongated by up to two turns (Figure 5C–D). Finally, the N-terminally attached SBL closes upon actual cofactor and inhibitor binding.

Figure 5
Cofactor and inhibitor binding mechanism

During this process, the ASL is visible inside the ligand-binding pocket (Figure 5D) and SBL-2 adopts an entirely α-helical conformation (Figure 5B). Subsequently, SBL-2 is split into the 310-helices η2 and η3 and adopts a more open conformation (Figure 5D,G). A comparison with the ternary complex structure TCL-2 shows that a backbone flip of Ala95 propagates the movements of adjacent residues and leads to the subsequent closure of SBL-2 (Figure 5G). This peptide flip may be induced by the ordering of Ser197 located on the SBL, which allows the formation of a water-mediated hydrogen bond interaction with the carbonyl oxygen of Ala95. Simultaneously, the movement of Phe96 creates space for the binding of the TCL Bring and establishes an elongated acyl-channel. During NADP+ binding, a similar peptide flip mechanism was observed for the NADPH-dependent β-ketoacyl-ACP reductase ecFabG, which also belongs to the SDR family of proteins (Price et al., 2004).

We speculate that the concerted closure of both SBLs, instead of just the classical SBL, is the conformational change corresponding to the slow step of the slow-binding inhibition (Lu and Tonge, 2008). Based on sequence similarities, this mechanism should mainly be feasible for Staphylococci, several Bacilli and some Listeria strains since they contain a serine at position 197, which connects the SBLs (Figure 5G). Most other Gram-negative and Gram-positive bacteria harbor an alanine at this position and, thus, lack the required hydrogen bond acceptor (Figure 3). Consistently, all structurally characterized FabIs from Gram-negative organisms contain just a single flexible SBL (PDB codes: 2JJY, 2P91, 2WYU, 3EK2, 3GRK, 3K2E), as opposed to Bacillus FabIs (3OIF, 3OJE) and the related proteins bsFabL (3OIC) and ecFabG (1I01) which all feature three disordered or rearranged regions in their apo structure.

Dimer - tetramer transition

The apo FabI structures from Gram-negative bacteria contain homo-tetramers, whereas bcFabI/baFabI, in the absence of cofactor and inhibitor, were reported to be dimers in the crystal and in solution (Kim et al., 2010). So far, reports regarding the oligomeric state of saFabI have been inconsistent. It was noted that the published apo structures of saFabI (3GNS, 3GNT) are comprised of dimers, though the enzyme was found to be a tetramer in solution (Kim et al., 2010; Priyadarshi et al., 2010). Therefore two questions arose: What is the oligomeric state of saFabI prior to ligand binding? If dimers are present, does tetramerization and active site formation occur in a concerted fashion with cofactor and inhibitor binding as hypothesized for bsFabL (Kim et al., 2011)?

Our results clearly show that NADP+ and triclosan binding to dimeric saFabI induces simultaneous active site formation and tetramerization. In contrast to a previous report but in accordance with the respective apo saFabI structures (3GNS, 3GNT) (Priyadarshi et al., 2010), we identified a dimer through analytical size exclusion chromatography (SEC) experiments at pH 8.0 (Figure S3A), whereas tetramers are present at pH 5.6 (see Experimental Procedures for details). We performed further analytical SEC experiments with the dimeric enzyme in its apo form or incubated with NADP+, NADPH or NADP+ and TCL. Our results unambiguously demonstrate a transition between dimers and tetramers upon both inhibitor and cofactor binding (Figure S3B), whereas binding of oxidized or reduced cofactor alone is not sufficient to induce tetramerization.

A structure and interface analysis of all available saFabI structures provides snapshots of the dimer - tetramer transition in the order 3GNT, 3GNS, apo-1, apo-2, TCL-2 (Figure 6 and Movie S2). During this process, the buried surface area of the QR-interface, about which the association occurs, increases significantly, as does the number of hydrogen bonds and salt bridges between interface residues (Figure 6C). The QR-interface is established via a 4-helix bundle comprising the long helices α4/α5 (kinked) and α6 (Figure 6, last column). Additionally, the corresponding N-terminal extensions η3 and η4 further stabilize this interface (Figure 3). Since the SBL-2 and ASL regions including these 310-helices are disordered in our apo structures, the QR-interface is destabilized and the dimers move slightly (~2 Å) away from one another. During the ordering of the ASL, the full tetramer interface and the catalytic active site are simultaneously established (Figure S4). The 3GNS structure clearly contains dimers due to the additionally disordered α4 region (Figure 3). This helix is therefore essential for the maintenance of the QR-interface. Whereas helix α6 of 3GNS is almost completely structured and just slightly shifted, the N-terminal portion of this helix is unstructured and partially disordered in 3GNT.

Figure 6
Dimer - tetramer transition


We hypothesize that the dimer - tetramer transition also applies to substrate binding, representing the molecular basis for the previously reported positive cooperative binding of NADPH to saFabI (Heath et al., 2000). Our data clearly show that NADPH binds cooperatively to saFabI with an approximate Hill coefficient h = 2 (Figure S5). Additionally, trans-2-octenoyl-CoA bound cooperatively to saFabI with a Hill coefficient h = 2.3. However, glutamate, which enhances the catalytic activity of the enzyme (Supplemental Discussion), was found to alter the cooperativity of binding to saFabI. In the presence of 1 M potassium glutamate, no cooperativity was observed for the binding of NADPH or trans-2-octenoyl-CoA to saFabI. Since cooperativity was still observed in the presence of 250 mM potassium glutamate, which is an upper limit estimate of the average S. aureus intracellular glutamate concentration (Björklind and Arvidson, 1978; Blanche et al., 1996), it is likely to be physiologically relevant. Mechanistically, the observed positive cooperativity of substrate binding could be transferred via the ligand-interacting SBL-2 and ASL regions and the attached long helices α4 and α6, which primarily constitute the QR-interface. The formation of such an interface would enhance cofactor and inhibitor/substrate binding due to the rearrangement of the two loop regions and vice versa.

Cooperativity has, to our knowledge, never been observed for NADH-dependent wild-type FabI enzymes, which already exist as tetramers in their apo forms (Heath et al., 2001). In contrast, a similar FabI tetramerization process might be possible for Bacillus species as α4 is additionally disordered in the dimeric bcFabI/baFabI apo structure (PDB code: 3OJE:A) (Kim et al., 2010). Similarly, the twisted tetramer architecture of the bsFabL apo structure (3OIC:A) comprises N-terminally shifted α4 and α6-helices and therefore links this protein to saFabI (Kim et al., 2011).

Cofactor specificity

The studies described above reveal fundamental differences between saFabI and classical FabI proteins. Hence, we also investigated cofactor specificity since, in contrast to all Gram-negative FabI homologues studied thus far, saFabI prefers NADPH instead of NADH as the reducing agent (Heath et al., 2000; White et al., 2005). Our structural analysis confirmed that the positively charged side chains of Arg40 and Lys41 are important for binding the additional 2′-phosphate of NADPH (Priyadarshi et al., 2010; Xu et al., 2008) (Figure 7A). However, we identified an RKXXS-motif that confers this unique specificity to saFabI. While the R40Q/K41N saFabI double mutant exhibited a drastically decreased kcat/Km value for NADPH, the specificity constant remained low for NADH (Xu et al., 2008). Our structures revealed Ser44 as a third crucial residue for NADPH specificity. The serine hydroxyl group forms a hydrogen bond to one of the phosphate oxygen atoms at a distance of 2.9 ± 0.1 Å, while the Ser44-bound phosphate oxygen forms additional hydrogen bonding interactions with the backbone amide nitrogens of Arg40 and Lys41, which are located in a tight loop between β2 and α2 (Figure 7A).

Figure 7
Structural basis of NADPH specificity

Unlike saFabI, the enoyl-ACP reductase from E. coli (ecFabI) is NADH-dependent (Stewart et al., 1999). Superposition of saFabI with ecFabI shows that Leu44 and Gln40 clash with the 2′-phosphate of a hypothetically bound NADPH (shortest distances of 1.7 ± 0.1 and 1.5 ± 0.2 Å, Figure 7B). Bulky substituents at position 44 (numbers according to saFabI), such as Leu or Phe (Figure 3), appear to shrink the adenine ribose-binding pocket, thus decreasing the affinity towards NADPH.

To test the hypothesis that the RKXXS-motif is responsible for cofactor specificity, we generated a triple mutant with all three phosphate-interacting amino acids replaced by the respective E. coli residues (R40Q/K41N/S44L). Interestingly, compared to results reported in the absence of glutamate (Xu et al., 2008), there is only a 2.5-fold increase in the specificity constant of wild-type saFabI for NADPH relative to NADH in the presence of glutamate (Table 2). The glutamate-containing conditions also strongly affected the specificity constant of the R40Q/K41N mutant for NADH, which recovered to that of the wild-type enzyme for NADPH. Additionally, the specificity constant of the double mutant for NADH was 200-fold higher than for NADPH, compared to the 19-fold difference reported in the absence of glutamate. The triple mutant displayed an even more dramatic inversion of cofactor specificity with the specificity constant for NADPH nearly 10,000-fold lower than for NADH. This was driven mostly by a decrease in the ability to utilize NADPH, consistent with our prediction that Leu44 would clash with the 2′-phosphate. Interestingly, the Km,NADPH of wild-type saFabI is higher than the Km,NADH of the triple mutant (Tables 2 and S3) and other FabI homologues (Parikh et al., 1999; Sivaraman et al., 2003) which correlates well with the estimated higher microbial intracellular pool of NADPH compared to NADH (Bennett et al., 2009; Liebeke et al., 2010).

Table 2
Kinetic parameters for saFabI wild-type, double and triple mutant utilizing NADPH (top) and NADH (bottom) at saturating trans-2-octenoyl-CoA concentrations (150 μM).

Using BLAST and PHI-BLAST searches (Altschul et al., 1990) with the newly identified RKXXS-motif, we found that this preference for NADPH links saFabI to all Staphylococcus and some Bacillus FabIs, as well as FabL and FabG, rather than to classical NADH-dependent FabIs.

Branched-chain substrate specificity

What is the underlying biological significance of the structural variations we observed for the FabIs from Staphylococci and Bacilli? Notably, the membranes of Staphylococcus and Bacillus genera contain branched-chain fatty acids (BCFA) as a major component (Kaneda, 1991). In contrast, most other bacteria (Kaneda, 1991) predominantly synthesize straight-chain fatty acids (SCFA). For the more common SCFA family members, membrane fluidity is controlled by unsaturated fatty acids (UFA), whereas, for BCFA family members, 12-methyltetradecanoic acid (anteiso-C15) primarily fulfills this purpose (Kaneda, 1991). Thus, we speculated that the increased mobility of FabI from Staphylococci and Bacilli allows the preferential binding of branched-chain substrates.

In addition to different straight-chain substrates (see Supplemental Results), we analyzed two branched-chain substrate analogues - trans-5-methyl-2-hexenoyl-CoA and (±)-trans-4-methyl-2-hexenoyl-CoA, which contains the precursor analog to the anteiso fatty acids found predominantly in the S. aureus membrane (Parsons et al., 2011). Among the first-round substrates (Figure 1), the ratio of specificity constants is approximately 1:24:1 (straight = trans-2-butenoyl-CoA: iso: anteiso) (Table 3), although the substrate specificity of the anteiso-substrate may be higher depending on the stereospecificity of the enzyme. Considering that FabI is the rate-limiting enzyme in the FAS II pathway (Heath and Rock, 1995), this ratio is likely to be physiologically significant in determining the composition of fatty acids synthesized and incorporated into the cell membrane. Moreover, since the pool of first-round substrates entering the FAS II cycle is generated by the branched-chain specific S. aureus FabH enzyme, the synthesis of BCFAs should be highly favored in this pathway (see Supplemental Results for further details) (Qiu et al., 2005). To substantiate the relevance of this observation, we explored the branched-chain substrate specificity of the NADH-dependent FabI enzyme from the Gram-negative pathogen F. tularensis (ftFabI). The ratio of specificity constants (Table S3) for the shortest chain substrates is approximately 800:72:1 (straight: iso: anteiso) and hence fundamentally different from saFabI.

Table 3
Kinetic parameters for wild-type saFabI utilizing various trans-2-enoyl-CoA substrates. See also Figure S1 and Table S3.


Our combined results clearly indicate that saFabI possesses distinct characteristics that markedly differentiate it from classical FabI enzymes, while exhibiting similarities to Staphylococcus and Bacillus FabIs and FabL. We identified an RKXXS-motif, which is responsible for the preferential binding of NADPH to saFabI and most likely to FabI proteins of other Staphylococci and some Bacillus species. Our analysis also reveals a fundamental extension of the classical inhibitor binding mechanism including conformational changes in three regions and a dimer - tetramer transition upon cofactor and inhibitor binding that may also be applicable for FabI enzymes of related Staphylococcus and Bacillus species. The oligomeric transition is coupled to ligand binding and hence may provide the mechanistic underpinnings for the observed cooperativity upon substrate binding. Glutamate was shown to influence cofactor differentiation and to increase the activity of saFabI while reducing the cooperativity of substrate binding. Nevertheless, cooperativity was still observed at a physiologically relevant glutamate concentration, suggesting that glutamate may function as an intracellular metabolite that regulates enzyme activity (for further details see Supplemental Discussion and Figure S5).

In contrast to many other bacteria including Streptococci, the cell membranes of Staphylococcus and Bacillus species mainly comprise branched-chain fatty acids (Balemans et al., 2010; Brinster et al., 2009; Fozo and Quivey, 2004; Kaneda, 1991; O’Donnell et al., 1985; Parsons et al., 2011). We propose that S. aureus FabI, as well as Bacillus FabI and FabL proteins, have evolved differently compared to classical FabI proteins to enable the production of BCFAs. The additional flexibility observed in the saFabI structures and other characterized Bacillus FabIs may play a role in altering the substrate specificity to accommodate the more bulky branched-chain substrates. Accordingly, Ser197 in saFabI, which links the two flexible substrate binding loops after ligand binding (Figure 5G), is conserved in Staphylococci, several Bacilli and some Listeria. Furthermore, we found that FabI enzymes capable of utilizing NADPH belong mostly to the BCFA family of organisms, however, the rationale behind this observation is currently not clear. To generate a membrane containing mostly branched-chain fatty acids, S. aureus and B. subtilis FabH enzymes are known to prefer branched-chain acyl-CoA primers instead of acetyl-CoA (Figure 1, Supplemental Results) (Choi et al., 2000; Qiu et al., 2005). Our data clearly show that this preference is carried on in the FAS II cycle as the substrate specificity for the first-round branched-chain substrates relative to the first-round straight-chain substrate is markedly increased for saFabI compared to FabIs from organisms producing mainly SCFAs.

Since BCFAs are known to be important for the in vivo fitness of S. aureus and Listeria monocytogenes (Singh et al., 2008; Sun and O’Riordan, 2010), the requirement of some organisms for these fatty acids may explain the different susceptibility of Gram-positive bacteria to inhibitors of the fatty acid biosynthesis pathway. Importantly, only a minimal amount of BCFAs are present in the human and murine blood (1% of all plasma fatty acids in humans (Holman et al., 1995) and less than 1% in mice (Atshaves et al., 2005; Gloerich et al., 2005)). It is known that the modulation of membrane lipids enables bacteria to survive under distinct stress situations (Singh et al., 2008). Even though both types of fatty acids serve to increase the fluidity of the membrane bilayer, the structural and morphological membrane characteristics differ for BCFAs and UFAs (Legendre et al., 1980). Hence, compared to UFAs, supplementing with BCFAs did not confer the same level of fitness to E. coli UFA auxotrophs in response to cold stress (Silbert et al., 1973). Similarly, other evidence suggests that S. aureus and L. monocytogenes fitness is reduced in the absence of BCFAs. The survival of branched-chain α-ketoacid dehydrogenase (BKD) deficient mutant strains in murine animal models was significantly reduced (Singh et al., 2008; Sun and O’Riordan, 2010). Nevertheless, future experiments should clarify whether the defined composition of the human blood lipid pool, comprising mainly SCFAs and UFAs, can fulfill the in vivo survival and virulence requirements of Staphylococci and other BCFA family members (Brinster et al., 2009; Holman et al., 1995).

As the essentiality of the FAS II pathway for S. aureus infection in vivo is re-validated, a detailed structural and kinetic characterization of targets within the pathway becomes increasingly relevant. FabI is one such target, which has spurred many inhibitor discovery efforts. We have shown that the drug target FabI in S. aureus and closely related pathogens differs from its homologous proteins with respect to cofactor and substrate specificity, cofactor/inhibitor binding and quaternary structure. Knowledge of these differences, as well as the structures solved here and in similar studies, can aid in the development of antibiotics specifically targeting FabI enzymes from the important human pathogens S. aureus, S. epidermidis, B. anthracis and B. cereus, which all synthesize branched-chain fatty acids. Remarkably, three saFabI inhibitors are in phase I clinical trials since 2009, providing substantial hope for new MRSA drugs (Gerusz, 2010).

Experimental Procedures

Cloning, expression and purification

We optimized the saFabI purification protocol with respect to plasmid usage, E. coli expression strain, cultivation and buffer composition (see Supplemental Experimental Procedures for further details) based on previously described procedures (Priyadarshi et al., 2010; Xu et al., 2008). A detailed description of the protocols is provided in Table S4. FtFabI was prepared as described previously (Lu et al., 2009).

Site-directed mutagenesis, expression and purification of saFabI mutants

Site-directed mutagenesis was performed using the QuikChange Mutagenesis Kit from Stratagene to add a single point mutation (S44L) to the previously constructed saFabI R40Q/K41N mutant (primers: 5′-ACCAGAACGAACGTCTGCGTAAAGAGCTTGAA-3′ and 5′-TTCAAGCTCTTTACGCAGACGTTCGTTCTGGT-3′) (Xu et al., 2008). The sequence of saFabI R40Q/K41N/S44L was confirmed by DNA sequencing. Expression and purification of the double and triple mutant was performed as described in Table S4.

Crystallization, data collection and structure determination

Prior to co-crystallization utilizing the vapor diffusion method, purified saFabI was incubated for 2 h at 4 °C with a 10-fold molar excess of NADP+ (100 mg/ml stock solution in water) and a 20-fold molar excess of the respective inhibitor supplemented as solid powder or dissolved in DMSO (for the P1 structures, 100 mg/ml stock solutions), respectively. The complexes were crystallized by the hanging drop vapor diffusion method using different precipitants (see Supplemental Experimental Procedures). Diffraction data of the flash-frozen crystals were collected at BESSY II (Berlin) or the ESRF (Grenoble), respectively. All structures were solved by molecular replacement with baFabI (2QIO) as the initial search model (see Supplemental Experimental Procedures and Tables S1 and S2 for further details).

Analytical size exclusion chromatography

SaFabI was dialyzed into a buffer containing 20 mM Tris pH 8.0 and 200 mM NaCl. Equal sample volumes were applied to a calibrated 10/300 Superdex 200 GL column (GE Healthcare) pre-equilibrated with dialysis buffer. Prior to analytical SEC the protein (5 mg/ml) was supplemented with a 10-fold molar excess of NADP+, NADPH or an equal volume of water and a 20-fold molar excess of TCL (100 mg/ml stock solution in DMSO) or an equal volume of DMSO, respectively. All four samples (apo, NADP+, NADPH and NADP+/TCL) were incubated at 20 °C for 2 h. Experiments were performed in triplicate and the peak shift confirmed for a different protein batch at pH 8.0 (Figure S3B). Additionally, one saFabI sample was analyzed at pH 5.6 in 20 mM trisodium citrate pH 5.6, 500 mM NaCl and 100 mM EDTA (Figure S3A).

Synthesis of trans-2-enoyl-CoA substrates

Trans-5-methyl-2-hexenoic acid and (±)-trans-4-methyl-2-hexenoic acid were synthesized from isovaleraldehyde (Sigma-Aldrich) and (±)-2-methylbutyraldehyde (Sigma-Aldrich), respectively, via a Horner-Wadsworth-Emmons reaction with methyl (triphenylphosphoranylidene)acetate, as described previously (Ryzhkov, 1996). Trans-2-enoyl-CoA substrates were synthesized from their respective trans-2-enoic acid using the mixed anhydride method (Parikh et al., 1999). Products were characterized by electrospray ionization mass spectrometry. Trans-2-butenoyl-CoA was purchased from Sigma-Aldrich.

Steady-state kinetic assays

Kinetic experiments were performed on a Cary 100 spectrophotometer (Varian) at 20°C in 50 mM potassium phosphate, 150 mM NaCl and 1 M potassium glutamate pH 7.5 containing 8% glycerol (v/v) and 0.1 mg/mL bovine serum albumin (BSA). Reaction velocities were measured by monitoring the oxidation of NAD(P)H to NAD(P)+ at 340 nm (ε = 6220 M−1 cm−1). Kinetic parameters for saFabI were determined by measuring initial velocities at varying concentrations of one substrate, holding the other substrate concentration constant (for further details see Supplemental Experimental Procedures).

Inhibition kinetics

Progress curves were used to determine the steady-state inhibition of saFabI by the slow-onset inhibitors TCL, EPP and CPP. The final reaction mixture contained saFabI (100 nM), trans-2-butenoyl-CoA (1.5 mM), NADPH (350 μM), NADP+ (400 μM) and inhibitor (2% v/v DMSO). Ki*,app values, corresponding to the final steady-state inhibition following slow isomerization of the initial enzyme-inhibitor complex, were determined by plotting the fractional steady-state velocities as a function of inhibitor concentration and fitting to the standard isotherm equation (Equation 1).


where vu is the initial velocity of the uninhibited reaction (for further details see Supplemental Experimental Procedures).


  • Insights into saFabI ligand binding including a dimer-tetramer transition
  • Identification of a loop motif that determines altered cofactor specificity
  • Increased flexibility modulates substrate and inhibitor recognition
  • Ability of saFabI to reduce branched-chain fatty acid precursor molecules

Supplementary Material





We thank the staff at the ESRF beamlines ID 14-4 and ID 29 (Grenoble) and at the BESSY II beamline 14.1 (Berlin) for technical support. This work was supported in part by NIH grants AI044639 and AI070383 to P.J.T., and through the Deutsche Forschungsgemeinschaft to C.K. (SFB630 and Forschungszentrum FZ82). J.S. was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. A.C. was supported by the Medical Scientist Training Program (MSTP, NIH T32GM008444) and by the Chemical Biology Training Program (CBTP, NIH T32GM092714).


Accession numbers

The coordinates and structure factors of saFabI in its unliganded form (space groups P32 and P43212) as well as in complex with NADP+ and triclosan (space groups P212121 and P1), CPP or EPP have been deposited in the PDB with the codes 4ALN, 4ALM, 4ALL, 4ALI, 4ALJ and 4ALK, respectively.

The authors declare that no conflict of interest exists.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. [PubMed]
  • Atshaves BP, McIntosh AL, Payne HR, Mackie J, Kier AB, Schroeder F. Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene. Am J Physiol Cell Physiol. 2005;288:C543–558. [PubMed]
  • Balemans W, Lounis N, Gilissen R, Guillemont J, Simmen K, Andries K, Koul A. Essentiality of FASII pathway for Staphylococcus aureus. Nature. 2010;463:E3. discussion E4. [PubMed]
  • Bamber AI, Neal TJ. An assessment of triclosan susceptibility in methicillin-resistant and methicillin-sensitive Staphylococcus aureus. J Hosp Infect. 1999;41:107–109. [PubMed]
  • Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol. 2009;5:593–599. [PMC free article] [PubMed]
  • Bissantz C, Kuhn B, Stahl M. A medicinal chemist’s guide to molecular interactions. J Med Chem. 2010;53:5061–5084. [PMC free article] [PubMed]
  • Björklind A, Arvidson S. Influence of Amino Acids on the Synthesis of an Extracellular Proteinase from Staphylococcus aureus. Journal of General Microbiology. 1978;107:367–375.
  • Blanche F, Cameron B, Bernard FX, Maton L, Manse B, Ferrero L, Ratet N, Lecoq C, Goniot A, Bisch D, Crouzet J. Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases. Antimicrob Agents Chemother. 1996;40:2714–2720. [PMC free article] [PubMed]
  • Brinster S, Lamberet G, Staels B, Trieu-Cuot P, Gruss A, Poyart C. Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens. Nature. 2009;458:83–86. [PubMed]
  • Choi KH, Heath RJ, Rock CO. beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol. 2000;182:365–370. [PMC free article] [PubMed]
  • Escaich S, Prouvensier L, Saccomani M, Durant L, Oxoby M, Gerusz V, Moreau F, Vongsouthi V, Maher K, Morrissey I, Soulama-Mouze C. The MUT056399 inhibitor of FabI is a new antistaphylococcal compound. Antimicrob Agents Chemother 2011 [PMC free article] [PubMed]
  • Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science. 2009;325:1089–1093. [PMC free article] [PubMed]
  • Fozo EM, Quivey RG., Jr Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl Environ Microbiol. 2004;70:929–936. [PMC free article] [PubMed]
  • Gerusz V. Recent Advances in the Inhibition of Bacterial Fatty Acid Biosynthesis. In: John EM, editor. Annual Reports in Medicinal Chemistry. Academic Press; 2010. pp. 295–311.
  • Gloerich J, van Vlies N, Jansen GA, Denis S, Ruiter JP, van Werkhoven MA, Duran M, Vaz FM, Wanders RJ, Ferdinandusse S. A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways. J Lipid Res. 2005;46:716–726. [PubMed]
  • Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics. 1999;15:305–308. [PubMed]
  • Grimm C, Maser E, Mobus E, Klebe G, Reuter K, Ficner R. The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family. J Biol Chem. 2000;275:41333–41339. [PubMed]
  • Heath RJ, Li J, Roland GE, Rock CO. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. J Biol Chem. 2000;275:4654–4659. [PubMed]
  • Heath RJ, Rock CO. Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli. J Biol Chem. 1995;270:26538–26542. [PubMed]
  • Heath RJ, White SW, Rock CO. Lipid biosynthesis as a target for antibacterial agents. Prog Lipid Res. 2001;40:467–497. [PubMed]
  • Holman RT, Adams CE, Nelson RA, Grater SJ, Jaskiewicz JA, Johnson SB, Erdman JW., Jr Patients with anorexia nervosa demonstrate deficiencies of selected essential fatty acids, compensatory changes in nonessential fatty acids and decreased fluidity of plasma lipids. J Nutr. 1995;125:901–907. [PubMed]
  • Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22:2577–2637. [PubMed]
  • Kaneda T. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev. 1991;55:288–302. [PMC free article] [PubMed]
  • Kim KH, Ha BH, Kim SJ, Hong SK, Hwang KY, Kim EE. Crystal Structures of Enoyl-ACP Reductases I (FabI) and III (FabL) from B. subtilis. J Mol Biol. 2011;406:403–415. [PubMed]
  • Kim SJ, Ha BH, Kim KH, Hong SK, Shin KJ, Suh SW, Kim EE. Dimeric and tetrameric forms of enoyl-acyl carrier protein reductase from Bacillus cereus. Biochem Biophys Res Commun. 2010;400:517–522. [PubMed]
  • Kleywegt GJ, Jones TA. Phi/psi-chology: Ramachandran revisited. Structure. 1996;4:1395–1400. [PubMed]
  • Krissinel E. Crystal contacts as nature’s docking solutions. J Comput Chem. 2010;31:133–143. [PubMed]
  • Krissinel E, Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr. 2004;60:2256–2268. [PubMed]
  • Legendre S, Letellier L, Shechter E. Influence of lipids with branched-chain fatty acids on the physical, morphological and functional properties of Escherichia coli cytoplasmic membrane. Biochim Biophys Acta. 1980;602:491–505. [PubMed]
  • Liebeke M, Meyer H, Donat S, Ohlsen K, Lalk M. A metabolomic view of Staphylococcus aureus and its ser/thr kinase and phosphatase deletion mutants: involvement in cell wall biosynthesis. Chem Biol. 2010;17:820–830. [PubMed]
  • Lu H, England K, am Ende C, Truglio JJ, Luckner S, Reddy BG, Marlenee NL, Knudson SE, Knudson DL, Bowen RA, et al. Slow-onset inhibition of the FabI enoyl reductase from francisella tularensis: residence time and in vivo activity. ACS Chem Biol. 2009;4:221–231. [PMC free article] [PubMed]
  • Lu H, Tonge PJ. Inhibitors of FabI, an enzyme drug target in the bacterial fatty acid biosynthesis pathway. Acc Chem Res. 2008;41:11–20. [PubMed]
  • Madsen SM, Beck HC, Ravn P, Vrang A, Hansen AM, Israelsen H. Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme. Appl Environ Microbiol. 2002;68:4007–4014. [PMC free article] [PubMed]
  • Maity K, Banerjee T, Prabakaran N, Surolia N, Surolia A, Suguna K. Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from plasmodium falciparum. IUBMB Life. 2011;63:30–41. [PubMed]
  • Mendoza D, Schujman GE, Aguilar PS. Biosynthesis and Function of Membrane Lipids. In: Sonenshein AL, Hoch JA, Losick R, editors. Bacillus subtilis and its closest relatives. Washington, DC: American Society for Microbiology; 2002. pp. 43–55.
  • O’Donnell AG, Nahaie MR, Goodfellow M, Minnikin DE, Hajek V. Numerical analysis of fatty acid profiles in the identification of staphylococci. J Gen Microbiol. 1985;131:2023–2033. [PubMed]
  • Pantosti A, Venditti M. What is MRSA? Eur Respir J. 2009;34:1190–1196. [PubMed]
  • Parikh S, Moynihan DP, Xiao G, Tonge PJ. Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis. Biochemistry. 1999;38:13623–13634. [PubMed]
  • Park HS, Yoon YM, Jung SJ, Yun IN, Kim CM, Kim JM, Kwak JH. CG400462, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor. Int J Antimicrob Agents. 2007;30:446–451. [PubMed]
  • Parsons JB, Frank MW, Subramanian C, Saenkham P, Rock CO. Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors. Proc Natl Acad Sci U S A. 2011;108:15378–15383. [PubMed]
  • Payne DJ, Miller WH, Berry V, Brosky J, Burgess WJ, Chen E, DeWolf WE, Jr, Fosberry AP, Greenwood R, Head MS, et al. Discovery of a novel and potent class of FabI-directed antibacterial agents. Antimicrob Agents Chemother. 2002;46:3118–3124. [PMC free article] [PubMed]
  • Payne DJ, Warren PV, Holmes DJ, Ji Y, Lonsdale JT. Bacterial fatty-acid biosynthesis: a genomics-driven target for antibacterial drug discovery. Drug Discov Today. 2001;6:537–544. [PubMed]
  • Price AC, Zhang YM, Rock CO, White SW. Cofactor-induced conformational rearrangements establish a catalytically competent active site and a proton relay conduit in FabG. Structure. 2004;12:417–428. [PubMed]
  • Priyadarshi A, Kim EE, Hwang KY. Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex with NADP and triclosan. Proteins. 2010;78:480–486. [PubMed]
  • Qiu X, Choudhry AE, Janson CA, Grooms M, Daines RA, Lonsdale JT, Khandekar SS. Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus. Protein Sci. 2005;14:2087–2094. [PubMed]
  • Ryzhkov LR. Radical Nature of Pathways to Alkene and Ester from Thermal Decomposition of Primary Alkyl Diacyl Peroxide. J Org Chem. 1996;61:2801–2808. [PubMed]
  • Sievert DM, Rudrik JT, Patel JB, McDonald LC, Wilkins MJ, Hageman JC. Vancomycin-resistant Staphylococcus aureus in the United States, 2002–2006. Clin Infect Dis. 2008;46:668–674. [PubMed]
  • Silbert DF, Ladenson RC, Honegger JL. The unsaturated fatty acid requirement in Escherichia coli. Temperature dependence and total replacement by branched-chain fatty acids. Biochim Biophys Acta. 1973;311:349–361. [PubMed]
  • Singh VK, Hattangady DS, Giotis ES, Singh AK, Chamberlain NR, Stuart MK, Wilkinson BJ. Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses. Appl Environ Microbiol. 2008;74:5882–5890. [PMC free article] [PubMed]
  • Sivaraman S, Zwahlen J, Bell AF, Hedstrom L, Tonge PJ. Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan: kinetic analysis of mutant FabIs. Biochemistry. 2003;42:4406–4413. [PubMed]
  • Stewart MJ, Parikh S, Xiao G, Tonge PJ, Kisker C. Structural basis and mechanism of enoyl reductase inhibition by triclosan. J Mol Biol. 1999;290:859–865. [PubMed]
  • Sun Y, O’Riordan MX. Branched-chain fatty acids promote Listeria monocytogenes intracellular infection and virulence. Infect Immun. 2010;78:4667–4673. [PMC free article] [PubMed]
  • Tipparaju SK, Mulhearn DC, Klein GM, Chen Y, Tapadar S, Bishop MH, Yang S, Chen J, Ghassemi M, Santarsiero BD, et al. Design and synthesis of aryl ether inhibitors of the Bacillus anthracis enoyl-ACP reductase. ChemMedChem. 2008;3:1250–1268. [PMC free article] [PubMed]
  • White SW, Zheng J, Zhang YM, Rock The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem. 2005;74:791–831. [PubMed]
  • Xu H, Sullivan TJ, Sekiguchi J, Kirikae T, Ojima I, Stratton CF, Mao W, Rock FL, Alley MR, Johnson F, et al. Mechanism and inhibition of saFabI, the enoyl reductase from Staphylococcus aureus. Biochemistry. 2008;47:4228–4236. [PMC free article] [PubMed]