Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2007 Feb;16(2):261–272. doi: 10.1110/ps.062473707

Structure of the human β-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

Caspar Elo Christensen 1,2, Birthe B Kragelund 1, Penny von Wettstein-Knowles 1, Anette Henriksen 2
PMCID: PMC2203288  PMID: 17242430

Abstract

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The β-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the α-methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C2 fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C16 and C18 products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C6 and C10–C12) substrate preferences leading to the C8 lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding.

Keywords: fatty acid synthesis, X-ray crystal structure, hexanoyl, acyl binding, antibiotics, binding pockets, cerulenin

Fatty acid biosynthesis was long thought to be differently organized in diverse organisms. The type I fatty acid synthase (FAS) complexes present in the cytoplasm of mammals, fungi, and lower eukaryotes are multifunctional proteins encoded by a single gene or, in some cases, two genes. Type II FAS present in prokaryotes and plant plastids consists of discrete enzymes. Prokaryotic fatty acid biosynthesis is being regarded as an obvious target for novel antibiotics (Campbell and Cronan 2001; Heath et al. 2001; Zhang et al. 2006b). Triclosan, which inhibits the enoyl reductase of type II FAS, is used in everyday applications such as toothpaste, sportswear, and kitchen utensils (Bhargava and Leonard 1996; Heath et al. 1998, 1999). The mammalian cytoplasmic type I FAS is the presumed target for the inhibitors cerulenin and C75, inducing apoptosis in some cancer cell lines and stopping weight gain in mice (Kuhajda et al. 2000; Loftus et al. 2000; Haber et al. 2003; Zhou et al. 2003; Alli et al. 2005). Initial evidence was obtained in the early 1990s for a mitochondrial type II FAS (Mikolajczyk and Brody 1990; Harington et al. 1993). These results were extended and have led to the recent cloning and characterization of the remaining mitochondrial FAS components from a range of organisms including yeast, humans, and Arabidopsis thaliana (Zhang et al. 2003; Kastaniotis et al. 2004; Yasuno et al. 2004), thus establishing the existence of a type II FAS in mitochondria. Its presence complicates the search for novel antibiotics as well as the interpretation of the effect of FAS inhibition in eukaryotic systems once thought to contain only the cytoplasmic FAS.

In prokaryotic FAS, the target of cerulenin, C75, thiolactomycin, and the recently reported inhibitor platensimycin is the condensing enzyme β-ketoacyl [acyl carrier protein] synthase (KAS) (D'Agnolo et al. 1973; Omura 1976; Nishida et al. 1986; Kauppinen et al. 1988; Kuhajda et al. 2000; Wang et al. 2006). KAS enzymes catalyze the Claisen condensation of a primer acyl chain with malonyl-acyl carrier protein (ACP) converting the primer to a β-ketoacyl-ACP elongated by a C2 unit. The reaction mechanism is tripartite. (1) First, an ACP-activated primer acyl substrate accesses the buried active site through a funnel. Here it is trans-thioesterified to the active-site cysteine and the acyl chain inserted in the hydrophobic acyl-binding pocket. Then, (2) the donor malonyl-ACP accesses the active site through the same funnel entering the malonyl-binding pocket. Its decarboxylation generates a reactive C2-carbanion (3) that attacks and condenses with the α-carbon of the bound primer, resulting in its release as β-ketoacyl bound to ACP. The position of the active-site cysteine at the N-terminal end of an α-helix lowers its pK a value, enabling trans-thioesterification. The decarboxylation reaction is accelerated through either primer binding, binding of inert substrates, or mutation of the active-site cysteine (Kresze et al. 1977b; Witkowski et al. 1999; McGuire et al. 2001). Three KAS isozymes with different substrate and inhibitor specificities are present in Escherichia coli—EcKASI, EcKASII, and EcKASIII. EcKASIII catalyzes the initial condensation reaction of acetyl-CoA and malonyl-ACP, while EcKASI and EcKASII both elongate saturated fatty acids up to C16. EcKASI is essential for the initiation of unsaturated fatty acid synthesis (Garwin et al. 1980a), while EcKASII alone catalyzes the last elongation in unsaturated fatty acid biosynthesis, palmitoleic (C16:1) to cis-vaccenic (C18:1) acid (Rosenfeld et al. 1973; D'Agnolo et al. 1975; Garwin et al. 1980b). In the absence of acetyl-CoA, EcKASI decarboxylation produces acetyl-ACP (McGuire et al. 2001), while little or none is detected using EcKASII and Streptococcus pneumoniae KASII (von Wettstein-Knowles et al. 2000; Zhang et al. 2006a). EcKASI is highly sensitive toward cerulenin, EcKASII moderately sensitive, and EcKASIII insensitive (D'Agnolo et al. 1975; Garwin et al. 1980a; Jackowski and Rock 1987; Edwards et al. 1997; McGuire et al. 2000). Human and yeast type I FASes are also sensitive to cerulenin, while thiolactomycin is FAS type II specific (Vance et al. 1972; D'Agnolo et al. 1973; Hayashi et al. 1983; Nishida et al. 1986; Jackowski et al. 1989). The three KAS isozymes constitute two groups; KASI + II have a Cys-His-His (CHH) catalytic triad, while KASIII has a Cys-His-Asn (CHN). The KAS activity of type I FASes and the mitochondrial KASes belongs to the CHH group. Crystallization of acyl-KAS complexes has failed to unveil the background of the different substrate specificities (Olsen et al. 2001).

The human mitochondrial KAS (HsmtKAS) was cloned and characterized by Zhang et al. (2005). Like the A. thaliana mitochondrial KAS (AtmtKAS) (Yasuno et al. 2004), HsmtKAS includes an N-terminal mitochondrial target peptide. HsmtKAS is less sensitive to cerulenin than other CHH enzymes and has a distinct bimodal substrate specificity (Zhang et al. 2005). To add to the understanding of KAS enzymes and to support the development of new antibiotics and anti-oncogenic FAS inhibitors specific for either mitochondrial or cytoplasmic KAS, we present the X-ray crystal structures of HsmtKAS and of the covalent hexanoyl complexes of HsmtKAS (HsmtKAS:C6) and AtmtKAS (AtmtKAS:C6). The structures shed light on rearrangements taking place in the active site upon substrate binding and on structural features important for inhibitor binding and substrate specificity differences between KASI and II enzymes.

Results

The structures of HsmtKAS and enzyme:acyl complexes

HsmtKAS was purified and its activity confirmed by decarboxylation assays (data not shown). Crystallographic analysis revealed that HsmtKAS is a dimer with the well-known α-β-α-β-α-thiolase fold first described by Mathieu et al. (1994) capped by an α-helical region connecting the strands of the N-terminal β-sheet (Fig. 1). Dimerization buries 3455 Å2, similar to the case of AtmtKAS (Olsen et al. 2004), but 200–1200 Å2 more than in the bacterial KAS enzymes. Whereas an extra α-helix in the cap region (Fig. 2B) contributed to the interface enlargement in AtmtKAS, an extra two-stranded N-terminal β-sheet and the burying of pockets that form the interface enlargement do so in HsmtKAS. The overall architecture is remarkably conserved. AtmtKAS and EcKASII are the closest structural neighbors to HsmtKAS (normalized RMSD100 = 0.6 Å for 399/394 Cα positions) (Carugo and Pongor 2001); EcKASI comes next (0.8 Å, 380 Cα), and finally EcKASIII (1.27 Å, 198 Cα). A loop (Pro93–Phe101) connecting the first and fourth strands of the N-terminal β-sheet is unique to the mitochondrial KAS enzymes (Fig. 2A). Despite the structural similarity, a significant difference is the lack of the α-helical insertion characterizing AtmtKAS, which is replaced by a loop, Gly78–Ile84, resembling that of the KASI and II enzymes (Fig. 2A).

Figure 1.

Figure 1.

Open in a new tab

(A) Alignment of relevant sequence segments of KAS enzymes, numbered as in HsmtKAS. Residues bordering the HsmtKAS acyl-binding pocket are marked with the letter “a,” while those bordering the connecting tunnel are marked with the letter “c.” Shading refers to the degree of conservation. The sequences are AtmtKAS (Yasuno et al. 2004), EcKASI (Kauppinen et al. 1988), EcKASII (Edwards et al. 1997), Saccharomyces cerevisiae mtKAS (ScmtKAS) (Harington et al. 1993), S. pneumoniae KASII (SpKASII) (Price et al. 2003), Synechocystis sp. KASII (SspKASII) (Moche et al. 2001), and the human KAS domain of type I FAS (HsFAS) (Jayakumar et al. 1995). (B) Cartoon representation of HsmtKAS:C6 dimer in stereoview. The acyl chain is shown in orange spheres and selected residues in red sticks: the active-site residues Cys209, His348, His385, Phe447, and the switch Met154. One subunit is colored light gray and labeled with blue amino acid residue numbers, the other is colored according to the structural regions of the protein. The N- and C-terminal regions are blue and yellow, respectively, except for the α-helical extrusions forming the cap region, which are green. The two NH4 + ions (cyan) present below the active sites are represented as spheres. The illustration was prepared in PyMol (DeLano 2002).

Figure 2.

Figure 2.

Open in a new tab

Cα RMSDs between (A) HsmtKAS and HsmtKAS:C6 and (B) AtmtKAS and AtmtKAS:C6. The tube diameter is proportional to the RMSD between the structures. The extreme RMSD values are 0.01 and 1.66 Å in HsmtKAS-HsmtKAS:C6 and 0.01 and 2.32 in AtmtKAS-AtmtKAS:C6. The colors, sticks, and spheres are as in Figure 1. (C,D) Plots of the Cα RMSDs of HsmtKAS and AtmtKAS subunits A and B, respectively. Schematic drawings of the secondary structural elements bar with color coding as in Figure 1 are inserted underneath the plots.

The crystal structures of the hexanoyl complexes do not deviate significantly from the structures of the free enzymes (RMSD = 0.5 and 0.3 Å between 3023 HsmtKAS and 3222 AtmtKAS atoms, respectively). The major deviations between HsmtKAS and HsmtKAS:C6 are located in the cap region as illustrated by the tube diameters of the Cα RMSD comparison of Figure 2A and the green color in the secondary structure bar indicator in Figure 2C. Apart from the extreme RMSD value in the 424–427 loop at the surface of subunit B, little structural change is seen in the AtmtKAS–AtmtKAS:C6 comparison (Fig. 2B,D); nevertheless, also in this case the largest deviations are found in the cap region. The overall lower RMSD level observed between the AtmtKAS structures compared to the HsmtKAS structures might be a result of differences in crystal packing interactions.

Acyl-binding-induced rearrangements in the active site

The active-site catalytic CHH triad is located within a 5 Å radius at the bottom of a 15 Å deep water-lined funnel with a molecular volume of 1260 Å3 (Fig. 3A, red balls and sticks close to purple surface). [All volumes given in the text are molecular (Connolly's) volumes calculated with CASTp (Binkowski et al. 2003).] The overall architecture of the active site resembles other characterized CHH enzyme structures (Fig. 4). Upon formation of the acyl–enzyme complex, the active site subtly rearranges. In the complex the Cζ of Phe447 moves 1.25 Å relative to its position in the HsmtKAS structure (Figs. 3B, 4A), and the phenyl residue is fixed with the plane of the aromatic ring parallel to the bound acyl chain, 3.7 Å from the ligand C2 atom (Figs. 3C, 4A). The Phe447 B-factors in the complex decrease by ∼50% to the level of the surrounding residues.

Figure 3.

Figure 3.

Open in a new tab

Cavities close to the Met154 switch in the acyl-binding pocket of HsmtKAS. (A) Overall view of the cavities present in HsmtKAS. The active-site access funnel is in purple, the acyl-binding pocket plus active-site residues are in red, Met154 is in cyan, the water-free extension plus Phe248 is in green, and the water-filled connecting tunnel joining the Met154s plus Leu184 is in blue. The backbone traces of subunits A and B are included in cartoon representation in light and dark gray. (B,C) Close ups of the cavities in HsmtKAS and HsmtKAS:C6, respectively. (D) A modeling of HsmtKAS:C6 with Met154 shown in a rotamer allowing access to the water-free extension. (E) The acyl-binding pocket of EcKASI:cerulenin complex (PDB code: 1FJ8) (Price et al. 2001a). The surfaces are colored as above. Cerulenin is included in balls and sticks inside the acyl-binding pocket. The catalytic triad is included in red balls and sticks, and Met197 occupies the water-free extension; Gln113 and Glu200 are in gray sticks. All surfaces and cavities were calculated with a 1.3 Å probe in MSMS (Sanner et al. 1996), and the illustration was prepared with Dino3D (Philippsen 2002) and Povray. (F) Cartoon showing the acyl-binding pocket rearrangements necessary for long chain substrate binding.

Figure 4.

Figure 4.

Open in a new tab

Stereoviews of the HsmtKAS active site. (A) C6 in the acyl-binding pocket. Balls and sticks are colored according to atom type and origin: C atoms from HsmtKAS and the HsmtKAS:C6 are green and light blue, respectively, N atoms, dark blue; O atoms, red; and S atoms, yellow. Selected water molecules are included as red (HsmtKAS) and blue (HsmtKAS:C6) spheres. The F o − F c map, calculated excluding the C6 atoms from the model, is contoured at 3σ. Atoms within hydrogen bond distance of each other are connected with dotted lines, colored blue in the HsmtKAS structure and green in the complex. The shortest distance between the ligand and Met154 is included in black. (B) Model of cerulenin from the EcKASII:cerulenin complex (PDB code: 1B3N) (Moche et al. 1999) (red sticks) and the EcKASI:cerulenin complex (PDB code: 1FJ8) (Price et al. 2001a) (orange sticks) made by superimposing the cerulenin containing structures on the HsmtKAS:C6 structure. The illustration was prepared in PyMOL.

In HsmtKAS, a high B-factor water molecule, W1, is located above the active-site Cys209 (Fig. 4A, center). This particular water molecule is conserved throughout known KAS structures, and under scrutiny for playing a role in decarboxylation as a nucleophile activated by His348 (Zhang et al. 2006a). In HsmtKAS, W1 is within hydrogen bond distance of Phe445 O and His348 Nɛ (Fig. 4A, center left, dashed green lines). In the HsmtKAS:C6 complex, W1 is displaced 0.7 Å toward His385 Nɛ entering hydrogen bond distance of this atom and breaking the hydrogen bond with the backbone O of Phe445 (Fig. 4A, dashed blue lines). The W1 B-factor drops to the level of the surroundings, and a network of ordered water molecules appears above the active site. One of these water molecules bridges the broken bond between Phe445 and W1 (Fig. 4A, center left, blue sphere). Another water molecule, W2, situated on the other side of His348 moves into hydrogen bond distance of the Nɛ of this residue (Fig. 4A, lower center, blue sphere and dashed blue line).

In the AtmtKAS:C6 complex, significant side-chain rearrangements in the active site are not observed, and only small adjustments in the active-site water structure follow formation of the thioester bond. Whereas W2 movement is different in AtmtKAS:C6 and the E. coli KASI and II complexes, the adjustments in the W1 position are consistent with the water structure adjustment mentioned above (Olsen et al. 2001; von Wettstein-Knowles et al. 2006). The B-factors of the AtmtKAS Phe447 analog shift likewise.

A switch in the acyl-binding pocket

In HsmtKAS:C6 and AtmtKAS:C6, electron densities extending from the active-site Cys209s into the cap were modeled as hexanoyl trans-thioesterized to the active-site Cys (Fig. 4A, F o − F c omit map, gray mesh). In the uncomplexed forms buried, hydrophobic-residue-lined cavities are present from Cys209 to Met154 in HsmtKAS and to Ile154 in AtmtKAS (Met/Ile154) (Fig. 1A; Fig. 3A,B, acyl binding pocket, red; Fig. 4B; Table 1). They are 10 Å long with molecular volumes of ∼115 Å3.

Table 1.

Amino acid composition and lengths of intramolecular pockets in HsmtKAS

graphic file with name 261tbl1.jpg

Open in a new tab

The cavities are not solvent accessible prior to ligand binding because Phe447 blocks the entrance (Fig. 3A,B). Asp/Glu237 is situated at the end of the acyl-binding pockets, but does not contribute to the pockets’ hydrophilicity as the carboxyl O is hydrogen bonded to the backbone Ns of Gly388 and Gly153 (not included in the figure). In the C6 complexes, the acyl chains fill the pocket causing minimal distortion (Figs. 3C, 4A). A maximum of one extra C atom is allowed in the pocket without reorientation of Met/Ile154. Separate from the acyl-binding pocket immediately beyond Met/Ile154, two as yet undescribed cavities exist in HsmtKAS and AtmtKAS (Fig. 3A,B). (1) Met/Ile154 and Leu184 restrict access to a water-lined cavity crossing the dimer interface and connecting to Met/Ile154 of the other subunit (Figs. 3A,B, connecting tunnel, blue). (2) Met/Ile154 and a small water-free hydrophobic cavity (Figs. 3A,B, water-free extension, green) exist immediately above and behind Met/Ile154 and Phe248 (Figs. 3A,B, 4B). Upon C6 ligand binding, the same cavities exist, except that the water-free extension is only found in one of the HsmtKAS:C6 subunits because of a difference in Met154 orientation between the subunits. The orientation of the neighboring Cys239 residue also differs between the subunits of HsmtKAS, indicating flexibility in the region (Fig. 3B,C). A scanning of optimal rotamers of Met154 in HsmtKAS:C6 and Ile154 in AtmtKAS:C6 shows that the acyl-binding pocket can be extended into the water-free extension by a rotation in the Met/Ile154 χ-angles enlarging the pocket to a total of 220 Å3 (Figs. 3D, red surface). In addition to the water-free extension, the increased volume represents the volume previously occupied by Met154 and Phe447. The extended acyl-binding pocket has room for C12 substrates. None of the Met/Ile154 rotamers in either HsmtKAS or AtmtKAS allowed access from the acyl-binding pocket to the connecting tunnel. On the basis of these characteristics, we postulate that Met/Ile154 functions as a switch.

Discussion

The framework for the decarboxylation reaction

Acyl chain elongation by the Claisen condensation reaction mechanism includes generation of a carbanion from malonyl by decarboxylation (von Wettstein-Knowles et al. 2000). That His385 promotes decarboxylation by hydrogen bond donation from Nɛ is well established (Olsen et al. 2001; von Wettstein-Knowles et al. 2006; Zhang et al. 2006a). The electronic state of His348 and its part in decarboxylation remain enigmatic. Upon acylation of the active-site cysteinyl, the side chain of Phe447 in HsmtKAS changes from poorly defined to a fixed rotamer, and W1 is fixed within hydrogen bond distance of both histidine Nɛs. The rearrangements taking place indicate a possible change in the state of His348. Primer binding to the active-site cysteinyl accelerates the decarboxylation reaction, even with inert primers such as iodoacetamide (Kresze et al. 1977a). The same is true when the active-site cysteinyl has been mutated to Gln or Ala (Witkowski et al. 1999; McGuire et al. 2001), suggesting that the change most likely originates in the active-site pocket propagating through the imidazole ring of His348. The positional adjustment and fixation of W1 directly connected to the active-site histidines in the KAS:acyl complexes reflect the rearrangement activating decarboxylation upon primer binding. The positional adjustment may result from the conversion of the hydrophilic active-site cysteinyl to a hydrophobic thioester, prompting a fixed network of structured water molecules to appear. Thus, when poised for decarboxylation, W1 is positioned immediately between the active site histidines, suggesting that both Nɛs interact with the same substrate atom, unlike the various mechanisms proposed by Olsen et al. (2001), Witkowski et al. (2002), and Zhang et al. (2006a). The accelerated production of the derailment product triacetic acid lactone (TAL) in S. pneumoniae KASII Phe447Ala, shows that Phe447 is not required for decarboxylation, as more substrate is being consumed in its absence. Instead, a role for Phe447 as inhibitor of decarboxylation in the situation where no primer is bound is more compatible with the results of Zhang et al. (2006a).

Acylation and its implications for inhibitor binding

With a molecular volume of ∼115 Å3 and a depth of 10 Å, the longest acyl chain fitting in the binding pocket without distorting Met154 is C7. This is far below the apparent maximum HsmtKAS substrate length of C14 (Zhang et al. 2005) and too little for binding of the C12 antibiotic cerulenin (Fig. 4B, orange and red, balls and sticks). In EcKASII, the pocket is equally small. The Met154 homolog Ile108 terminates the binding pocket, giving a volume of 71 Å3 (Huang et al. 1998). The crystal structure of the cerulenin–EcKASII complex reveals that a rearrangement of Ile108 takes place, expanding the pocket to 215 Å3, which is comparable to the modeled 220 Å3 extended acyl-binding pocket in HsmtKAS:C6 (Fig. 3D, red acyl-binding pocket; Moche et al. 1999). That the region is likely to harbor such rearrangements in HsmtKAS is indicated by torsion/libration/screw (TLS) analysis (Painter and Merritt 2006). TLS analysis allows the description of anisotropic, static disorder on a per segment basis. Using 20 segments, TLS analysis of HsmtKAS and HsmtKAS:C6 showed that in HsmtKAS:C6, the segments surrounding the hypothesized pocket extension add flexibility to this volume. The major changes taking place upon binding the C6 ligand in HsmtKAS localize to this part of the structure as well (Fig. 2A). Likely this extended pocket will be available for binding cerulenin and medium to long chain acyl substrates in HsmtKAS and AtmtKAS (Fig. 3F).

The energy barrier caused by the required rearrangement of Met154 explains the low C8-substrate preference (K cat/K m) observed by Zhang et al. (2005). With respect to the longer C10 and C12 substrates, the cost of rearranging Met154 is compensated by interaction with the highly hydrophobic hypothesized extension. Binding of substrates longer than C12, which have very low K cat/K m values (Zhang et al. 2005), is presumably impeded by their need to expand the pocket further. The higher sensitivity toward cerulenin of AtmtKAS (Olsen et al. 2004) than HsmtKAS (Zhang et al. 2005) may reflect the linear character of Met versus Ile. That the position occupied by Met154 plays a central role in substrate binding was first highlighted by a mutation study (Val et al. 2000), where Ile108Phe in EcKASII exhibited a dramatic reduction in elongation activity with substrates longer than C6.

Analysis of ligand-free and longer acyl chain ligand–KAS complexes shows that in EcKASI complexes [C12: PDB no. 1EK4 (Olsen et al. 2001); cerulenin: 1FJ8 (Price et al. 2001b)], the acyl-binding pocket extends toward the connecting tunnel (Figs. 3E, 4B), while in the KASII–cerulenin complex, the ligand extends toward the possible pocket extension identified in this study (Figs. 3F, 4B). In the KASI structure, a methionine positioned in the cap region, EcKASI Met197, occupies the water-free pocket extension, making it unavailable for substrate binding. KASII structures have no access to a pocket for long chain substrates resembling the part of the KASI acyl-binding pocket harboring the ω-end of long chain fatty acids. The combined action of Met154, Ile183, and Leu184 effectively obstructs the formation of a similar binding pocket in HsmtKAS. The active sites of the two subunits are not connected in available KASI structures, since the side chains of EcKASI Gln113 and Glu200 interact at the dimer interface (Fig. 3E). These observations indicate substantial differences between the acyl-binding pockets of KASI and KASII enzymes providing an explanation for the variations in substrate specificity between KAS I and II enzymes.

Superposition of the structures of HsmtKAS and the EcKASII–platensimycin complex (Wang et al. 2006) reveals that HsmtKAS should provide as good a target for platensimycin as EcKASII. In HsmtKAS:C6, Phe447 adopts the conformation allowing edge-to-face aromatic stacking suggested as central for platensimycin binding (Wang et al. 2006). Arg252 positioned at the beginning of the active-site access funnel is the sole potential impediment for ketolide binding. As Arg252 is disordered in HsmtKAS and found in two alternative conformations in HsmtKAS:C6, this is unlikely to cause problems. The malonyl-binding pocket, as defined in the EcKASI:thiolactomycin complex (Price et al. 2001a), is conserved in HsmtKAS (Pro317, Phe445, and Phe447).

The low degree of similarity between the residues lining the acyl-binding pocket in HsmtKAS and the human KAS domain of FAS (Fig. 1A) suggests that acyl tail modifications of cerulenin might provide potential targeting differences between HsmtKAS and human type I FAS. Tetrahydrocerulenin lacking double bonds in the acyl tail does not affect human type I FAS; nevertheless, it inhibits T24 cell proliferation (Lawrence et al. 1999). Tetrahydrocerulenin is active against EcKASII (D'Agnolo et al. 1973; Moche et al. 1999) and fungal type I FAS (Morisaki et al. 1993). Interestingly, the primary structure of the KAS domain of human type I FAS diverges from the KASII sequences in the region around M154, where all prokaryotic KASIIs have Ile and EcKASI has a Gly (Fig. 1A).

Conclusion

Likely, the state of the active-site histidines of KASI + II enzymes changes upon substrate binding. When binding acyl chains longer than C7, the acyl-binding pockets of HsmtKAS and AtmtKAS could be extended through a change in the rotamer of the Met/Ile154 switch residues. The rotation will extend the pockets into cavities similar to the one made available in EcKASII upon cerulenin binding. In EcKASI, this pocket is blocked by Met197, and long chain acyl substrates are passed toward a pocket similar to the connecting tunnel. The different potential pocket extensions are described here for the first time, and their presence offers an explanation for the different substrate specificities observed in KASI + II enzymes.

The coordinates and structure factors have been deposited in the Protein Data Bank at EBI (http://www.ebi.ac.uk/msd) (IDs: HsmtKAS, 2IWY; HsmtKAS:C6, 2IWZ; AtmtKAS:C6, 2IX4).

Materials and methods

Identification of the HsmtKAS

To identify a putative HsmtKAS, the expressed sequence tag (EST) database was searched with the sequence of EcKASII (Siggaard-Andersen et al. 1994). The sequence with the highest degree of similarity (GenBank accession no. AK000611.1) encoded a putative KAS. The primary sequence translation product was 51% identical to AtmtKAS, 46% identical to the EcKASII sequence, and 35% identical to EcKASI sequence. The catalytic CHH triad and residues assigned a role in the structure or reaction mechanism of KASI/KASII were all conserved in the sequence including the EcKASI Lys328 and Phe395. The primary sequence included a 38 amino acid N-terminal target sequence, predicted to target mitochondria by TargetP 1.0 (Emanuelsson et al. 2000).

Cloning, expression, and protein purification

A clone of AK000611 was kindly provided by Sumio Sugano, Tokyo University (Yudate et al. 2001). Using the forward primer 5′-GGTGGTGGTGGATCCATTGAGGGGCGCTCCAGATTGCATAGGC-3′ (having a BamHI restriction site in bold and coding for a factor Xa cleavage site) and the reverse primer 5′-GGTGGTGGTAAGCTTCTACAGTCCAGCAAT-3′ (HindIII site in bold), the coding sequence was ligated into pQE-30 (QIAGEN), substituting the 37-codon leader with an MRGS-HHHHHHGS-IEGR tag. The resultant plasmid was transformed into the E. coli XL-1 blue strain (Stratagene), verified by sequencing, and transformed into the E. coli expression strain M15. Protein was expressed and purified as previously described (McGuire et al. 2001), except that DTT was substituted with the more stable TCEP (0.2 mM), sonication was for five instead of three rounds, and induction was overnight at 20°C. The protein was further purified by anion exchange chromatography on a MonoQ HR 10/10 column (Amersham Biosciences) mounted on an ÄKTA Purifier HPLC system (Amersham Biosciences) with a pH 9 Tris-HCl buffer system [A buffer: 30 mM Tris at pH 9, 10 mM NaCl, 0.2 mM EDTA, 0.2 mM TCEP, and 8.7% (w/v) glycerol; B buffer: buffer A + 2 M NaCl]. After elution, the buffer was changed to storage buffer [150 mM NaCl, 30 mM Tris at pH 7.8, 2 mM EDTA, 0.2 mM TCEP 8.7% (w/v) glycerol], and the protein was concentrated to 4 mg/mL in an Amicon Ultra-15 filter device (Millipore) and stored at −20°C. Decarboxylation activity was verified in conformationally sensitive urea gels as carried out by McGuire et al. (2001). AtmtKAS was produced as described previously (Olsen et al. 2004).

Crystallization and data collection

Crystals of HsmtKAS and HsmtKAS:C6 were grown in hanging drops. Drops were composed of 2 μL of 87 μM protein solution in storage buffer and 2 μL of reservoir solution containing 24% (w/v) polyethylenglycol 3350 and 0.2 M NH4Cl. In the co-crystallization experiment, 4 mM hexanoyl-CoA was added to the protein solution prior to setting up the drops. After 5–8 d at room temperature, rod-shaped single crystals appeared. The crystals were flash cooled in N2(l). Data were collected at 100 K to 2 Å from the HsmtKAS crystal at beamline I711 (Cerenius et al. 2000), MAXlab, Lund University; and to 1.6 Å from the complex at beamline X11 at EMBL, Hamburg. High- and a low-resolution (3.5 Å cutoff) oscillation scans were collected from the HsmtKAS:C6 complex. The exposure time was halved, and the oscillation angle per frame was increased from 0.2° to 0.5° for the low-resolution scan. Data were integrated with MOSFLM and merged and scaled with SCALA (Evans 1997).

Attempts to soak acyl-CoA derivatives into the previously characterized AtmtKAS crystals (Olsen et al. 2004) failed. Screening of co-crystallization conditions resulted in plate-shaped single crystals when hanging drops were incubated at 22°C. Two microliters of a protein solution (5 mg/mL AtmtKAS, 20 mM bis-tris-buffer at pH 6.0, 200 mM KCl, 2 mM DTT) was mixed with 2 μL of 0.1 M MES (pH 6.5), 12% (w/v) PEG20000, and 1 μL of 50 mg/mL hexanoyl-CoA. The crystals were immersed in 35% (w/v) PEG4000, 0.1 M bis-tris (pH 6.5) for 5 sec and flash cooled in N2(l). Diffraction data to 1.95 Å resolution were collected at the X11 beamline, EMBL Hamburg.

Phasing and model building

The phases of the HsmtKAS structure factors were assigned by molecular replacement using the structure of AtmtKAS without water molecules (Olsen et al. 2004), as search model with MOLREP (Vagin and Teplyakov 1997). The model was built automatically using ArpWarp (Perrakis et al. 1999) with one homodimer in the asymmetric unit. The model was improved by hand building in O (Jones et al. 1991) and Coot (Emsley and Cowtan 2004), supplemented first by rigid-body refinement and simulated annealing in CNS (Brünger et al. 1998) and at later stages by positional refinement in REFMAC5 (Collaborative Computational Project, No.4 1994; Murshudov et al. 1997). Water molecules were added using the water picking procedure in CNS. One solvent molecule in each subunit's active site was refined as an NH4 + ion. The phases from the refined HsmtKAS model were used to solve the structure of the covalent HsmtKAS:C6 complex, which was refined as above except that a hexanoyl molecule was fitted into an excess electron density emerging from the active-site thiol. Water was included with Coot, and the Babinet method was used to model bulk solvent. To fully exploit the quality of the data sets, TLS anisotropic refinement was applied using tensors generated with the online TLSMD server (Schomaker and Trueblood 1968; Painter and Merritt 2006). The HsmtKAS structure was refined with five segments per subunit (residues A36–A158, A159–A284, A285–A320, A321–A431, A432–A459, B37–B130, B131–B239, B240–B312, B313–B430, B431–B459), and the complex with two segments per subunit (A30–A180, A181–A459, B35–B158, B159–B459). Only the last four residues of the engineered leader sequence were ordered and only in the A molecule of the HsmtKAS structure. In the B molecule, only the backbone and Cβ atom of the last residue of the engineered sequence could be traced. The orientation of a few residues and side chains could not be determined and were either left out (−) or included in truncated form—HsmtKAS: A (Arg252 Cα, Thr315-, Ala316-, Pro317-, Glu322 Cβ, Thr434-, Glu435-, Lys436-); HsmtKAS:C6: A (Glu98 Cβ, Lys433 Cβ, Thr434 Cα, Glu435 Cα, Lys436 Cα), B (Glu320 Cβ). The following residues occurred in alternative conformations: HsmtKAS A (Val337, Gln338); HsmtKAS:C6 A (Gln104, Lys109, Met130, Lys197, Ser244, Arg252, Val337), B (Arg39, Ser96, His365).

The structure of AtmtKAS could be solved by rigid-body refinement in REFMAC5, giving an asymmetric unit of one homodimer. The structure of the AtmtKAS:C6 complex was refined using REFMAC5 and Coot by first, manually rebuilding the protein main and side chains in Coot; second, including water molecules from the Coot interface; third, including ligand molecules from the Coot interface; and last, modeling the two cations identified in the structure. No NCS symmetry restrains were applied in the refinement. Models were refined with NH4 + ions, Mg2+ ions, Ca2+, and K+ ions with occupancy of 1. Only the K+ and Ca2+ ions gave satisfactory B factors with these settings, and ligand–cation distances (average O–cation distance = 2.8 Å) are in accordance with the final interpretation of the ions as K+ (Harding 2006). The side chain of Thr354 has been modeled in two alternate conformations. One TLS segment was defined for each subunit. TLS group parameters were refined in REFMAC5 after resetting the B factors to 20. The inclusion of TLS resulted only in a limited drop in the R free (0.5%).

The data collection and refinement statistics are given in Table 2.

Table 2.

Data collection, phasing, and refinement statistics

graphic file with name 261tbl2.jpg

Open in a new tab

Acknowledgments

The Danish National Science Research Council supported this project through DANSYNC and grants 21-01-0622 and 21-04-0604. Beam line scientists Yngve Cerenius (I711, MAXlab, Lund University) and Santosh Panjikar (EMBL X11 beam line at the DORIS storage ring, DESY, Hamburg) are acknowledged for technical assistance during data collection, as is Annette Kure Andreassen for protein purification and crystal preparation of AtmtKAS. We are grateful to Sumio Sugano, Tokyo University, for the AK00611 clone.

Footnotes

Reprint requests to: Anette Henriksen, Biostructure Group, Carlsberg Laboratory, Copenhagen, DK-2500, Denmark; e-mail: anette@crc.dk; fax: +45 3327 4708; Penny von Wettstein-Knowles, Department of Molecular Biology, University of Copenhagen, DK-2100, Denmark; e-mail: knowles@biobase.dk; fax: +45 3532 2128.

Abbreviations: ACP, acyl carrier protein; cerulenin, (2R,3S)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide; C75, tetrahydro-4-methylene-2-octyl-5-oxo-3-furancarboxylic acid; DTT, dithiothreitol; FAS, fatty acid synthase; KAS, β-ketoacyl [acyl carrier protein] synthase; mtKAS, mitochondrial KAS; RMSD, root-mean-square deviation; TCEP, tris(2-carboxyethyl)phosphine; thiolactomycin, 4-hydroxy-3,5-dimethyl-5-(2-methylbuta-1,3-dienyl)-5H-thiophen-2-one; TLS, translation/libration/screw; Tris-HCl, tris(hydroxymethyl)aminomethane hydrochloride.

References

  1. Alli, P.M., Pinn, M.L., Jaffee, E.M., McFadden, J.M., and Kuhajda, F.P. 2005. Fatty acid synthase inhibitors are chemopreventive for mammary cancer in neu-N transgenic mice. Oncogene 24: 39–46. [DOI] [PubMed] [Google Scholar]
  2. Bhargava, H.N. and Leonard, P.A. 1996. Triclosan: Application and safety. Am. J. Infect. Control 24: 209–218. [DOI] [PubMed] [Google Scholar]
  3. Binkowski, T.A., Naghibzadeh, S., and Liang, J. 2003. CASTp: Computed atlas of surface topography of proteins. Nucleic Acids Res. 31: 3352–3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., and Pannu, N.S., et al. 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54: 905–921. [DOI] [PubMed] [Google Scholar]
  5. Campbell, J.W. and Cronan, J.E. 2001. Bacterial fatty acid biosynthesis: Targets for antibacterial drug discovery. Annu. Rev. Microbiol. 55: 305–332. [DOI] [PubMed] [Google Scholar]
  6. Carugo, O. and Pongor, S. 2001. A normalized root-mean-square distance for comparing protein three-dimensional structures. Protein Sci. 10: 1470–1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cerenius, Y., Stahl, K., Svensson, L.A., Ursby, T., Oskarsson, A., Albertsson, J., and Liljas, A. 2000. The crystallography beamline I711 at MAX II. J. Synchrotron Radiat. 7: 203–208. [DOI] [PubMed] [Google Scholar]
  8. Collaborative Computational Project, No.4. 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763. [DOI] [PubMed] [Google Scholar]
  9. Cruickshank, D.W. 1999. Remarks about protein structure precision. Acta Crystallogr. D55: 583–601. [DOI] [PubMed] [Google Scholar]
  10. D'Agnolo, G., Rosenfeld, I.S., Awaya, J., Omura, S., and Vagelos, P.R. 1973. Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of β-ketoacyl-acyl carrier protein synthetase. Biochim. Biophys. Acta 326: 155–156. [DOI] [PubMed] [Google Scholar]
  11. D'Agnolo, G., Rosenfeld, I.S., and Vagelos, P.R. 1975. Multiple forms of β-ketoacyl-acyl carrier protein synthetase in Escherichia coli . J. Biol. Chem. 250: 5289–5294. [PubMed] [Google Scholar]
  12. DeLano, W.L. 2002. The PyMOL molecular graphics system. DeLano Scientific, San Carlos, CA.
  13. Edwards, P., Nelsen, J.S., Metz, J.G., and Dehesh, K. 1997. Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli . FEBS Lett. 402: 62–66. [DOI] [PubMed] [Google Scholar]
  14. Emanuelsson, O., Nielsen, H., Brunak, S., and Von Heijne, G. 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300: 1005–1016. [DOI] [PubMed] [Google Scholar]
  15. Emsley, P. and Cowtan, K. 2004. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol Crystallogr. 60: 2126–2132. [DOI] [PubMed] [Google Scholar]
  16. Evans, P.R. 1997. Scala. Jnt. CCP4 ESF–EACBM Newsl. on Protein Crystallogr. 33: 22–24. [Google Scholar]
  17. Garwin, J.L., Klages, A.L., and Cronan Jr., J.E. 1980a. Structural, enzymatic, and genetic studies of β-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli . J. Biol. Chem. 255: 11949–11956. [PubMed] [Google Scholar]
  18. Garwin, J.L., Klages, A.L., and Cronan Jr., J.E. 1980b. β-Ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J. Biol. Chem. 255: 3263–3265. [PubMed] [Google Scholar]
  19. Haber, E.P., Hirabara, S.M., Gomes, A.D., Curi, R., Carpinelli, A.R., and Carvalho, C.R.O. 2003. Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets. FEBS Lett. 544: 185–188. [DOI] [PubMed] [Google Scholar]
  20. Harding, M.M. 2006. Small revisions to predicted distances around metal sites in proteins. Acta Crystallogr. D. Biol. Crystallogr. 62: 678–682. [DOI] [PubMed] [Google Scholar]
  21. Harington, A., Herbert, C.J., Tung, B., Getz, G.S., and Slonimski, P.P. 1993. Identification of a new nuclear gene (CEM1) encoding a protein homologous to a β-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae . Mol. Microbiol. 9: 545–555. [DOI] [PubMed] [Google Scholar]
  22. Hayashi, T., Yamamoto, O., Sasaki, H., Kawaguchi, A., and Okazaki, H. 1983. Mechanism of action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of Escherichia coli . Biochem. Biophys. Res. Commun. 115: 1108–1113. [DOI] [PubMed] [Google Scholar]
  23. Heath, R.J., Yu, Y.T., Shapiro, M.A., Olson, E., and Rock, C.O. 1998. Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. J. Biol. Chem. 273: 30316–30320. [DOI] [PubMed] [Google Scholar]
  24. Heath, R.J., Rubin, J.R., Holland, D.R., Zhang, E., Snow, M.E., and Rock, C.O. 1999. Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J. Biol. Chem. 274: 11110–11114. [DOI] [PubMed] [Google Scholar]
  25. Heath, R.J., White, S.W., and Rock, C.O. 2001. Lipid biosynthesis as a target for antibacterial agents. Prog. Lipid Res. 40: 467–497. [DOI] [PubMed] [Google Scholar]
  26. Huang, W., Jia, J., Edwards, P., Dehesh, K., Schneider, G., and Lindqvist, Y. 1998. Crystal structure of β-ketoacyl-acyl carrier protein synthase II from E. coli reveals the molecular architecture of condensing enzymes. EMBO J. 17: 1183–1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jackowski, S. and Rock, C.O. 1987. Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria. J. Biol. Chem. 262: 7927–7931. [PubMed] [Google Scholar]
  28. Jackowski, S., Murphy, C.M., Cronan Jr., J.E., and Rock, C.O. 1989. Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin. J. Biol. Chem. 264: 7624–7629. [PubMed] [Google Scholar]
  29. Jayakumar, A., Tai, M.H., Huang, W.Y., al Feel, W., Hsu, M., Abu-Elheiga, L., Chirala, S.S., and Wakil, S.J. 1995. Human fatty acid synthase: Properties and molecular cloning. Proc. Natl. Acad. Sci. 92: 8695–8699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47: 110–119. [DOI] [PubMed] [Google Scholar]
  31. Kastaniotis, A.J., Autio, K.J., Sormunen, R.T., and Hiltunen, J.K. 2004. Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology. Mol. Microbiol. 53: 1407–1421. [DOI] [PubMed] [Google Scholar]
  32. Kauppinen, S., Siggaard-Andersen, M., and von Wettstein-Knowles, P. 1988. β-Ketoacyl-ACP synthase I of Escherichia coli: Nucleotide sequence of the fabB gene and identification of the cerulenin binding residue. Carlsberg Res. Commun. 53: 357–370. [DOI] [PubMed] [Google Scholar]
  33. Kresze, G.B., Steber, L., Oesterhelt, D., and Lynen, F. 1977a. Reaction of yeast fatty acid synthetase with iodoacetamide. 2. Identification of the amino acid residues reacting with iodoacetamide and primary structure of a peptide containing the peripheral sulfhydryl group. Eur. J. Biochem. 79: 181–190. [DOI] [PubMed] [Google Scholar]
  34. Kresze, G.B., Steber, L., Oesterhelt, D., and Lynen, F. 1977b. Reaction of yeast fatty acid synthetase with iodoacetamide. 3. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide. Eur. J. Biochem. 79: 191–199. [DOI] [PubMed] [Google Scholar]
  35. Kuhajda, F.P., Pizer, E.S., Li, J.N., Mani, N.S., Frehywot, G.L., and Townsend, C.A. 2000. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc. Natl. Acad. Sci. 97: 3450–3454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. 1993. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26: 283–291. [Google Scholar]
  37. Lawrence, D.S., Zilfou, J.T., and Smith, C.D. 1999. Structure-activity studies of cerulenin analogues as protein palmitoylation inhibitors. J. Med. Chem. 42: 4932–4941. [DOI] [PubMed] [Google Scholar]
  38. Loftus, T.M., Jaworsky, D.E., Frehywot, G.L., Townsend, C.A., Ronnett, G.V., Lane, M.D., and Kuhajda, F.P. 2000. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288: 2379–2381. [DOI] [PubMed] [Google Scholar]
  39. Mathieu, M., Zeelen, J.P., Pauptit, R.A., Erdmann, R., Kunau, W.H., and Wierenga, R.K. 1994. The 2.8 Å crystal structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: A five-layered α β α β α structure constructed from two core domains of identical topology. Structure 2: 797–808. [DOI] [PubMed] [Google Scholar]
  40. McGuire, K.A., McGuire, J.N., and von Wettstein-Knowles, P. 2000. Acyl carrier protein (ACP) inhibition and other differences between β-ketoacyl synthase (KAS) I and II. Biochem. Soc. Trans. 28: 607–610. [DOI] [PubMed] [Google Scholar]
  41. McGuire, K.A., Siggaard-Andersen, M., Bangera, M.G., Olsen, J.G., and von Wettstein-Knowles, P. 2001. β-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: Aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket. Biochemistry 40: 9836–9845. [DOI] [PubMed] [Google Scholar]
  42. Mikolajczyk, S. and Brody, S. 1990. De novo fatty acid synthesis mediated by acyl-carrier protein in Neurospora crassa mitochondria. Eur. J. Biochem. 187: 431–437. [DOI] [PubMed] [Google Scholar]
  43. Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. 1999. Structure of the complex between the antibiotic cerulenin and its target, β-ketoacyl-acyl carrier protein synthase. J. Biol. Chem. 274: 6031–6034. [DOI] [PubMed] [Google Scholar]
  44. Moche, M., Dehesh, K., Edwards, P., and Lindqvist, Y. 2001. The crystal structure of β-ketoacyl-acyl carrier protein synthase II from Synechocystis sp. at 1.54 Å resolution and its relationship to other condensing enzymes. J. Mol. Biol. 305: 491–503. [DOI] [PubMed] [Google Scholar]
  45. Morisaki, N., Funabashi, H., Shimazawa, R., Furukawa, J., Kawaguchi, A., Okuda, S., and Iwasaki, S. 1993. Effect of side-chain structure on inhibition of yeast fatty-acid synthase by cerulenin analogs. Eur. J. Biochem. 211: 111–115. [DOI] [PubMed] [Google Scholar]
  46. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53: 240–255. [DOI] [PubMed] [Google Scholar]
  47. Nishida, I., Kawaguchi, A., and Yamada, M. 1986. Effect of thiolactomycin on the individual enzymes of the fatty acid synthase system in Escherichia coli . J. Biochem. 99: 1447–1454. [DOI] [PubMed] [Google Scholar]
  48. Olsen, J.G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. 2001. Structures of β-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery. Structure 9: 233–243. [DOI] [PubMed] [Google Scholar]
  49. Olsen, J.G., Rasmussen, A.V., von Wettstein-Knowles, P., and Henriksen, A. 2004. Structure of the mitochondrial β-ketoacyl-[acyl carrier protein] synthase from Arabidopsis and its role in fatty acid synthesis. FEBS Lett. 577: 170–174. [DOI] [PubMed] [Google Scholar]
  50. Omura, S. 1976. The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis. Bacteriol. Rev. 40: 681–697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Painter, J. and Merritt, E.A. 2006. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62: 439–450. [DOI] [PubMed] [Google Scholar]
  52. Perrakis, A., Morris, R., and Lamzin, V.S. 1999. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6: 458–463. [DOI] [PubMed] [Google Scholar]
  53. Philippsen, A. 2002Dino: Visualizing structural biology.
  54. Price, A.C., Choi, K.H., Heath, R.J., Li, Z., White, S.W., and Rock, C.O. 2001a. Inhibition of β-ketoacyl-acyl carrier protein synthases by thiolactomycin and cerulenin. Structure and mechanism. J. Biol. Chem. 276: 6551–6559. [DOI] [PubMed] [Google Scholar]
  55. Price, A.C., Zhang, Y.M., Rock, C.O., and White, S.W. 2001b. Structure of β-ketoacyl-[acyl carrier protein] reductase from Escherichia coli: Negative cooperativity and its structural basis. Biochemistry 40: 12772–12781. [DOI] [PubMed] [Google Scholar]
  56. Price, A.C., Rock, C.O., and White, S.W. 2003. The 1.3-Å-resolution crystal structure of β-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae . J. Bacteriol. 185: 4136–4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Rosenfeld, I.S., D'Agnolo, G., and Vagelos, P.R. 1973. Synthesis of unsaturated fatty acids and the lesion in fab B mutants. J. Biol. Chem. 248: 2452–2460. [PubMed] [Google Scholar]
  58. Sanner, M.F., Olson, A.J., and Spehner, J.C. 1996. Reduced surface: An efficient way to compute molecular surfaces. Biopolymers 38: 305–320. [DOI] [PubMed] [Google Scholar]
  59. Schomaker, V. and Trueblood, K.N. 1968. On the rigid-body motion of molecules in crystals. Acta Crystallogr. B 24: 63–76. [Google Scholar]
  60. Siggaard-Andersen, M., Wissenbach, M., Chuck, J.A., Svendsen, I., Olsen, J.G., and von Wettstein-Knowles, P. 1994. The fabJ-encoded β-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates. Proc. Natl. Acad. Sci. 91: 11027–11031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vagin, A. and Teplyakov, A. 1997. MOLREP: An automated program for molecular replacement. J. Appl. Crystallogr. 30: 1022–1025. [Google Scholar]
  62. Val, D., Banu, G., Seshadri, K., Lindqvist, Y., and Dehesh, K. 2000. Re-engineering ketoacyl synthase specificity. Structure 8: 565–566. [DOI] [PubMed] [Google Scholar]
  63. Vance, D., Goldberg, I., and Mitsuhashi, O. 1972. Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem. Biophys. Res. Commun. 48: 649–656. [DOI] [PubMed] [Google Scholar]
  64. von Wettstein-Knowles, P., Olsen, J.G., McGuire, K.A., and Larsen, S. 2000. Molecular aspects of β-ketoacyl synthase (KAS) catalysis. Biochem. Soc. Trans. 28: 601–607. [DOI] [PubMed] [Google Scholar]
  65. von Wettstein-Knowles, P., Olsen, J.G., McGuire, K.A., and Henriksen, A. 2006. Fatty acid synthesis: Role of active site histidines and lysine in Cys-His-His-type β-ketoacyl-acyl carrier protein synthases. FEBS J. 273: 695–710. [DOI] [PubMed] [Google Scholar]
  66. Wang, J., Soisson, S.M., Young, K., Shoop, W., Kodali, S., Galgoci, A., Painter, R., Parthasarathy, G., Tang, Y.S., and Cummings, R., et al. 2006. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature 441: 358–361. [DOI] [PubMed] [Google Scholar]
  67. Witkowski, A., Joshi, A.K., Lindqvist, Y., and Smith, S. 1999. Conversion of a β-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine. Biochemistry 38: 11643–11650. [DOI] [PubMed] [Google Scholar]
  68. Witkowski, A., Joshi, A.K., and Smith, S. 2002. Mechanism of the β-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase. Biochemistry 41: 10877–10887. [DOI] [PubMed] [Google Scholar]
  69. Yasuno, R., von Wettstein-Knowles, P., and Wada, H. 2004. Identification and molecular characterization of the β-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase. J. Biol. Chem. 279: 8242–8251. [DOI] [PubMed] [Google Scholar]
  70. Yudate, H.T., Suwa, M., Irie, R., Matsui, H., Nishikawa, T., Nakamura, Y., Yamaguchi, D., Peng, Z.Z., Yamamoto, T., and Nagai, K., et al. 2001. HUNT: Launch of a full-length cDNA database from the Helix Research Institute. Nucleic Acids Res. 29: 185–188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Zhang, L., Joshi, A.K., and Smith, S. 2003. Cloning, expression, characterization, and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein. J. Biol. Chem. 278: 40067–40074. [DOI] [PubMed] [Google Scholar]
  72. Zhang, L., Joshi, A.K., Hofmann, J., Schweizer, E., and Smith, S. 2005. Cloning, expression, and characterization of the human mitochondrial β-ketoacyl synthase—Complementation of the yeast cem1 knock-out strain. J. Biol. Chem. 280: 12422–12429. [DOI] [PubMed] [Google Scholar]
  73. Zhang, Y.M., Hurlbert, J., White, S.W., and Rock, C.O. 2006a. Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae . J. Biol. Chem. 281: 17390–17399. [DOI] [PubMed] [Google Scholar]
  74. Zhang, Y.M., White, S.W., and Rock, C.O. 2006b. Inhibiting bacterial fatty acid synthesis. J. Biol. Chem. 281: 17541–17544. [DOI] [PubMed] [Google Scholar]
  75. Zhou, W., Simpson, P.J., McFadden, J.M., Townsend, C.A., Medghalchi, S.M., Vadlamudi, A., Pinn, M.L., Ronnett, G.V., and Kuhajda, F.P. 2003. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells. Cancer Res. 63: 7330–7337. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES