Structural and biochemical characterization of FabK from Thermotoga maritima

Biochemical and Biophysical Research Communications xxx (2016) 1e7

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Structural and biochemical characterization of FabK from Thermotoga maritima Byung Hak Ha a, 1, Sang Chul Shin a, c, 1, Jin Ho Moon a, Gyochang Keum b, Chan-Wha Kim c, Eunice EunKyeong Kim a, * a b c

Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea Brain Science Institute, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 November 2016 Accepted 25 November 2016 Available online xxx

TM0800 from Thermotoga maritima is one of the hypothetical proteins with unknown function. The crystal structure determined at 2.3 Å resolution reveals a two domain structure: the N-terminal domain forming a barrel and the C-terminal forming a lid. One FMN is bound between the two domains with the phosphate making intricate hydrogen bonds with protein and three tightly bound water molecules, and the isoalloxazine ring packed against the side chains of Met22 and Met276. The structure is almost identical to that of FabK (enoyl-acyl carrier protein (ACP) reductase, ENR II), a key enzyme in bacterial type II fatty-acid biosynthesis that catalyzes the final step in each elongation cycle; and the enzymatic activity confirms that TM0800 is an ENR. Enzymatic activity was almost completely abolished when the helices connecting the barrel and the lid were deleted. Also, the Met276Ala and Ser280Ala mutants showed a significant reduction in enzymatic activity. The crystal structure of Met276Ala mutant at 1.9 Å resolution showed an absence of FMN suggesting that FMN plays a role in catalysis, and Met276 is important in positioning FMN. TmFabK exists as a dimer in both solution and crystal. Together this study provides molecular basis for the catalytic activity of FabK. © 2016 Elsevier Inc. All rights reserved.

Keywords: FabK Enoyl-acyl carrier protein (ACP) reductase Crystal structure Biochemical characterization

1. Introduction Thermotoga maritima is a non-spore forming rod-shaped Gramnegative bacterium, which grows optimally at 80
C. This is the only organism known to live in such an extreme environment [1]. The genome of T. maritima MSB8 consists of a single circular 1.8-Mb chromosome encoding 1877 proteins, 1014 (54%) of which have functional assignments and 863 (46%) are of unknown function. Genome analysis shows that it shares 24% of its genome with members of Archaea. Because of its compact size, T. maritima has been considered as an ideal model for systems biology and metabolic reconstruction simulations. In addition, a campaign of highthroughput structure determination was carried out to define

Abbreviations: FabK, Enoyl-ACP reductase II; ENR, Enoyl-ACP reductase; FMN, Flavin mononucleotide; NADP, Nicotinamide adenine dinucleotide phosphate; PDB, Protein Data Bank; RMSD, root-mean-square deviation. * Corresponding author. E-mail address: [email protected] (E.E. Kim). 1 These authors contributed equally.

protein structure-function relation at the genomic scale [2]. TM0800 (SwissProt entry: Q9WZQ7) is one of the hypothetical proteins with molecular weight of 33,673 Da. It is annotated as a nitronate monooxygenase (NMO), formerly referred to as 2nitropropane dioxygenase, which is a FMN-dependent enzyme that uses molecular oxygen to oxidize alkyl nitronates [3e5]. It shares sequence identity of more than 50% with 2-nitropropane dioxygenase. The crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa (Pa2ND) shows a two domain structure, a TIM barrel connected to an inserted domain with a noncovalently bound FMN [4]. However, a BLAST search indicates that it also shares similar level of sequence identity with FabK, also known as enoyl-(acyl-carrier-protein)-reductase (ENR) II [6e8]. FabK catalyzes the final step of the fatty acid elongation cycle in the bacterial fatty acid biosynthesis (FAS II) pathway, i.e. the reduction of the double bond in trans-2-enoyl-ACP, in the same manner as FabI (also known as ENR I) which is more widely distributed ENR. Since ENR is a key enzyme, and broad spectrum antimicrobials such as diazaborine and triclosan inhibit ENR, they have been considered attractive targets for drug development [9]. The corresponding

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Please cite this article in press as: B.H. Ha, et al., Structural and biochemical characterization of FabK from Thermotoga maritima, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.141

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B.H. Ha et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7

Table 1 Statistics on data collection and refinement of TmFabK. MAD, Wild type

Data sets Beam line Wavelength (Å) Space group Unit cell parameters a (Å) b (Å) c (Å) a, b, g (
) Resolution range (Å) Total/unique reflections Completeness (%) I/s(I) Rmergeb (%) Refinement statistics Resolution range (Å) R/Rfree c (%) No. of protein atoms No. of water molecules No. of ligand molecules RMSD from ideal geometry: bond lengths (Å)/bond angles (
) MolProbity scored PDB entry a b c d

Met276Ala

Peak

Edge

Remote

KEK PF AR-NW12 0.9789 C2

0.9792

0.9814

65.724 77.180 59.172 90, 99.97, 90

65.797 77.213 59.230 90, 99.99, 90

228,464/12,928 91.5 (68.6) 30.1 (5.1) 7.0 (13.3)

232,136/12,995 87.3 (53.4) 27.8 (1.0) 4.7 (12.5)

65.611 77.107 59.085 90, 99.94, 90 50e2.3 (2.38e2.30)a 224,880/13,051 94.5 (77.0) 34.5 (6.7) 6.8 (11.7)

PAL 5C 1.0000 C2 84.096 115.129 67.913 90, 106.59, 90 50e1.9 (1.97e1.90) 1,472,459/48,755 96.3 (92.1) 39.5 (6.6) 4.2 (16.1)

40.4e2.3 19.3/23.0 2333 45 1

34.6e1.90 18.0/20.7 4414 271 2

0.007/0.820 1.71 5GVH

0.008/0.946 1.41 5GVJ

Values in parentheses are for the outer most resolution shell. P P P P Rmerge ¼ h irI (h,i)  r/ h i I (h,i), where I (h,i) is the intensity of the ith measurement of reflection h and is the mean value of I (h,i) for all i measurements. Rfree was calculated from randomly selected 10% set of reflections not included in the calculation of the R value. MolProbity score combines the clashcore, rotamer, and Ramachandran evaluation into a single score, normalized to be on the same scale as X-ray resolution.

reaction is also carried out by FabL (known as ENR III) [10] and FabV [11], and there are more than one ENR in some microbes. Interestingly, FabK from Streptococcus pneumoniae (SpFabK) also shows a TIM barrel structure with FMN bound at the active site [8] which is distinctly different from the structures of FabI, FabL [12,13] and FabV [14] which belong to the short-chain alcohol dehydrogenase reductase superfamily. Here we describe the structural and biochemical characterization of TM0800.

2. Materials and methods 2.1. Protein expression and purification TM0800 (SwissProt entry: Q9WZQ7) was amplified by PCR from T. maritima strain MSB8 genomic DNA, and the PCR product was cloned into Escherichia coli vector pET28a (Novagen) [15], and the primers used are shown in Supplementary Table S1. After transformation in BL21(DE3) (Stratagene), the cells were grown at 37
C until the optical density reached OD600 ¼ 0.6 and were then induced with 0.5 mM isopropyl-b-D-thiogalactopyranoside. The cells were lysed in 20 mM Tris at pH 8.0, 150 mM NaCl, 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 0.1 mM phenylmethylsulfonyl fluoride, and 50 mM NH4Cl by sonication and centrifugation. The supernatant after centrifugation was applied to a nickel-chelated Hi-Trap column (GE Healthcare) and eluted with a linear gradient of 25e500 mM imidazole in 20 mM Tris (pH 8.0) and 150 mM NaCl, 1 mM TCEP, 50 mM NH4Cl. The fractions collected were further purified using a Blue Sepharose Hi-Trap column (GE Healthcare) and eluted with a linear gradient of 0.1e2 M NaCl in 20 mM Tris (pH 8.0), 1 mM DTT, 50 mM NH4Cl. For the final step, size-exclusion chromatography on HiLoad 26/60 Superdex-75 column (GE Healthcare) pre-equilibrated with 20 mM Tris (pH 8.0), 300 mM NaCl, 1 mM DTT, 50 mM NH4Cl was carried

out. Since the structure of either NMO or FabK was not available when this structure was solved, selenomethionyl (SeMet) substituted protein was generated for multi-wavelength anomalous dispersion (MAD) phasing as described previously [16]. 2.2. Crystallization, data collection and processing Initial screening for the crystallization was carried out using a robotics system and the hits were further optimized using the hanging drop methods. The native crystals were grown under the conditions of 0.1 M Bicine, pH 9.0, 1.5 M LiCl and 23% (v/v) PEG 6000, while SeMet protein crystallized from a solution containing 50 mM sodium acetate, 1% (v/v) PEG 6000 and 45% (v/v) ethanol. Crystals belong to the space group C2 with a ¼ 65.611 Å, b ¼ 77.107 Å, and c ¼ 59.085 Å with b ¼ 99.94
. Both the native and the SeMet crystals were flash-frozen in liquid nitrogen prior to data collection at the Photon Factory (Tsukuba, Japan). Crystals of Met276Ala mutant were obtained by mixing equal volumes of ~30 mg/ml of protein in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 1 mM FMN with a reservoir solution containing 100 mM Tris-HCl (pH 8.0), 200 mM MgCl2, and 20% (v/v) PEG 20,000 in 2 days. Crystals belong to the same space group but with a ¼ 84.096 Å, b ¼ 115.129 Å, c ¼ 67.913 Å and b ¼ 106.59
with two molecules in the asymmetric unit. Diffraction data were collected at Pohang Light Source (Pohang, Korea). All diffraction data were processed using the program DENZO and SCALEPACK from HKL2000 [17]. Data statistics are summarized in Table 1. 2.3. Structure determination and refinement The structure was determined using the MAD phasing method. All possible Se sites were found and refined. The initial phases were calculated using the programs CCP4 suite [18], SOLVE [19] and RESOLVE [19] to a mean overall figure of merit of 0.59. An initial

Please cite this article in press as: B.H. Ha, et al., Structural and biochemical characterization of FabK from Thermotoga maritima, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.141

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Fig. 1. Overall structure of TmFabK. (A) Ribbon diagrams of TmFabK complexed with FMN shown in stick model. The electron density map (2FoFc) for the bound FMN and metal is contoured at 1s level. (B) Schematic diagram showing the interactions between FMN and TmFabK. (C) Interactions at the bound metal.

model was built using the ARP/wARP package [20], and COOT [21] and the final refinement was performed using the program CNS [22], REFMAC 5 [18] and PHENIX [23]. Since the native crystals did not diffract as well as the SeMet crystals, the SeMet data set was used for model building and refinement. The crystal structure of Met276Ala mutant was solved by molecular replacement method using the wild type and refined. Analysis of the stereochemical quality of the final model was confirmed using PROCHECK [24]. Figures were prepared using the program PyMOL [25]. The coordinates of both structures have been deposited in the Protein Data Bank, and final refinement statistics are summarized in Table 1. 2.4. Site-directed mutagenesis and activity assay The site-directed mutagenesis of residues of the lid at the FMN binding site, e.g. Tyr208, His229, Leu261, Met276 and Ser280 to Ala, were carried out. Also, the deletion mutants encompassing residues 208e215 (denoted as D208) and the helix 278e284 (denoted as D278) are made in the same manner (see Supplementary Table S1 for the primers used). The mutants were purified using the same procedure as the wild type. The ENR activity was monitored

spectrophotometrically based on the decrease in the absorbance at 340 nm using NADH at 25
C for 10 min using SpectraMAX 340 (Molecular Devices) using 10 mg TM0800 in 100 mM sodium phosphate at pH 7.6, 1 mM octyl-ACP and 1 mM NADH as described before [7]. 3. Results and discussion 3.1. Overall structure with bound FMN The crystal structure of TM0800 determined at 2.3 Å resolution was refined to R-values of 19.3% and 23.0% (Table 1). The overall structure consists of two domains: the N-terminal domain (residues 1e199) forming a (b/a)8 barrel (or triose phosphate isomerase barrel; TIM barrel) composed of eight stranded parallel beta sheet surrounded by eight alpha helices, and an additional ahelix formed by residues 289e312; the C-terminal domain (residues 200e288) forming a lid above the barrel (Fig. 1A). The lid consists of a b-hairpin packed against two a-helices. The lid is inserted between the 8th strand and the 8th helix of the barrel. The electron density maps, after placing all protein atoms, revealed clearly a bound FMN as shown in Fig. 1A. It is

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Fig. 2. Structural comparison. A) Structural comparison of TmFabK (this study PDB code: 5GVH), SpFabK (PDB code: 2Z6I), Pa2ND (PDB code: 2GJL), and SaNAO (PDB code: 3BW2) shown in green, pink, orange and light blue, respectively. (B) A close up at the FMN binding region. Residues involved in FMN binding are from four different regions, labelled as I-IV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

non-covalently bound in the deep pocket near the interface between the two domains, on the C-terminal side of the strands in the (b/a)8 barrel domain. The phosphate moiety of FMN is buried completely inside the pocket, held in place by intricate hydrogen bonds to the backbone amides (Gly172, Gly193 and Thr194) and the side chain atoms (Ser280, Thr194) as well as three tightly bound water molecules. On the other hand, the tricyclic isoalloxazine ring of FMN is partially accessible from the protein surface. It forms direct hydrogen bonds with the amide of Ala23 and the side chain of Asn71, and various contact interactions with the backbone and the side chain atoms as well as water molecules. The tricyclic ring is packed against the side chains of Met22 and Met276. The schematic representation of the various interactions is shown in Fig. 1B. There was another area of unaccounted electron density at the end of helices a10 and a14 (Fig. 1A), which was bigger than the typical water density. In addition, a cluster of carbonyl oxygen atoms are about 2.5 Å away from the center of the density, indicating that the density might be from a metal ion. Since the protein was crystallized in the presence of sodium, it was modeled accordingly, and the octahedral coordination around the metal is filled by a water molecule which is in turn hydrogen bonded to the carbonyl oxygen of Gly282 and the carboxyl oxygen of Asp285 (Fig. 1C). It appears that this metal might play a structural role, but it is not clear whether it plays any functional role. It has been reported earlier that the presence of a univalent metal or ammonium ion stimulates enzymatic activity [6,7]. 3.2. Structural comparison The TIM-barrel fold is one of the most common folds in the protein data bank and a number of enzyme families utilize this remarkably versatile scaffold to achieve appropriate active site geometry [26]. A DALI [27] search with the final model showed a number of significant matches, the closest being putative nitroalkan dioxygenase (TM0800) from T. maritima (PDB code: 3BO9), SpFabK [8], and FabK from Porphyromonas gingivalis (PDB code: 4IQL; [28]) with Z-scores ranging between 54 and 47 and rootmean-square deviation (RMSD) values of 0.6e1.6 Å. These were followed by Pa2ND [4] and nitroalkane oxidase from Streptomyces ansochromogenes (SaNAO; [29]) with Z-scores of 45 and 37 and RMSD of 1.4 and 2.0 Å, respectively. Another hit worth mentioning is enoyl reductase (ER) of the multifunctional fatty acid synthase

(FAS I) from fungus (PDB code: 2UVC; [30]) and yeast (PDB code:2PFF; [31]). FAS I is a multifunctional enzyme complex in which substrates are handed from one functional domain to the next, as opposed to the FAS II in which discrete enzyme activities are performed by discrete polypeptides encoded by discrete genes [32]. Fungi and vertebrates have FAS I for their de novo fatty acid synthesis whereas most bacteria and plants utilize FAS II. The ER showed Z-scores of about 30 and RMSD of around 2.5 Å for 300 Ca atoms with the sequence identity less than 20%. In the case of ER, the lid region is significantly larger (Supplementary Fig. S1). In fact, the helix a12 (250e258) of TM0800 is replaced by a helical domain composed of ~160 residues in ER, and NADPþ binds to this region (Supplementary Fig. S1B). Other proteins with TIM barrel fold such as inosine-5’-monophosphate dehydrogenase showed Z-scores of less than 22 despite sequence identities of 48e25%. The superposition of these structures showed that the N-terminal TIM barrel is almost identical while the C-terminal lid domains differs from one another (Fig. 2A). All except 3BO9 have a tightly bound FMN located at the interface between the two domains with the phosphate moiety anchored by intricate hydrogen bonds similar to what was described above. In the case of 3BO9, a phosphate is bound at the same position as the phosphate moiety of FMN making similar interactions with the protein atoms as well as water molecules. The orientation of FMN is about the same, but the interacting residues are different. The interacting residues are from four different parts of the molecule, and they are labelled I-IV as they appear in the sequence (Fig. 2B; Supplementary Fig. S2 for the sequence alignment). The regions I and IV are situated below and above FMN, e.g. the two methionines mentioned earlier in TM0800, namely Met22 and Met276, are from I and IV, respectively. While methionine below in the region I is conserved, the one in the region IV is replaced by a serine in Pa2ND and a tryptophan in SaNAO as seen in Fig. 2B. Interestingly, they are conserved within the family suggesting they may play a role in catalysis. On the other hand, the region II shows a larger variation in the length and composition, in particular it is significantly longer in SaNAO. The region III, corresponding to the loop connecting b6 and a7, contains the putative catalytic histidine, e.g. His146 in TM0800 is longer by two and eleven residues in Pa2ND and SaNAO, respectively. Nonetheless, the imidazole ring of the catalytic histidine lies above the isoalloxazine ring in all cases. Therefore, the structural comparison suggest that TM0800 is a FabK and not a nitronate

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Fig. 3. Mutational analysis and structure of Met276Ala. (A) Mutation sites. Tyr208, His229, Leu261, Met276 and Ser280 were mutated to Ala, while D208 and D278 denote deletion of residues 208e215 and 278e284, respectively. (B) Relative activities of the TmFabK mutants. Enzymatic activity was measured using the NADH-coupled assay. (C) Comparison of the wild type and Met276Ala mutant structures with a close up of the mutation site.

monooxygenase. It is worth noting that in the case of ER, the methionine in the region I is also conserved while it is a leucine in the region IV, and this leucine makes a direct contact with the nicotinamide moiety of NADPþ which is located just above FMN (Fig. S1) [30]. In all cases the average temperature factor for the lid region is much higher than that of the barrel, suggesting that the lid region has intrinsic flexibility, probably to accommodate the cofactors as well as the substrates. 3.3. Mutation analysis and structure of Met276Ala mutant ENR activity testing confirmed that TM0800 is a FabK, and hence should be named TmFabK. It showed selectivity for NADH over NADPH similar to SpFabK (data not shown). To investigate the role

5

Fig. 4. Dimeric TmFabK. (A) TmFabK forms a homodimer. Each molecule is indicated as A and A', since the two are crystallographically related in the wild type. (B) Dimer interface. Residues involved in the interactions are colored in light green on the molecular surface. The electrostatic surface of the interface is shown on the right. The view is rotated by 90
from that of (A). (C) Non-denaturing gel electrophoresis. Lane 1: BSA (66 and 132 kDa); Lane 2: Albumin (~45 kDa) as a protein marker (Sigma, USA), Lane 3: TmFabK wild type, Lane 4: TmFabK Met276Ala mutant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of the lid region on FMN binding and enzymatic activity, the two helices connecting the barrel and lid, namely a10 and a14, were deleted (Fig. 3A). Also mutated were the key residues involved in FMN binding which included Tyr208, Leu261 Met276 and Ser280, and His229 which is just above the catalytic His146 (Figs. 1B and 3A). They were mutated to an Ala and their relative activities are shown in Fig. 3B. The enzymatic activities of the two deletion

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mutants were almost abolished suggesting that the two helices are crucial for activity. Furthermore, while the mutations at Tyr209, His229 and Leu261 caused a slight reduction in catalytic activity, mutations at Met276 and Ser280 caused an approximately 50% reduction. These results suggest that the lid region of TmFabK contribute to catalytic activities e.g. substrate and cofactor binding. In particular, the Met276 and Ser280 mutants play a critical roles in the enzyme activity by supporting FMN binding as suggested from the structure. In order to confirm the results obtained by measuring enzymatic activity, the crystal structure of Met276Ala mutant was solved at 1.9 Å resolution. The overall structure is almost identical to that of the wild type with RMSD values of 0.38 Å and 0.49 Å for the two molecules in the asymmetric unit. However, in the Met271Ala structure residues 225e228 of both molecules, 246e256 of molecule A and 246e258 of molecule B were disordered. There was no electron density for FMN in both molecules. Instead, the side chain of Met22 occupies the FMN position in one molecule as shown in Fig. 3C. Together, these data suggest that Met276 is important in stabilizing FMN binding and presumably NAD(P)þ binding. 3.4. Dimer formation Although there is only one molecule in an asymmetric unit in the wild type, TmFabK exists as a tightly associated dimer with the second molecule generated by crystallographic 2-fold symmetry as shown in Fig. 4A. The two molecules are arranged such that the helices a7, a8 and a15 face each other at the dimer interface. The accessible surface area at the interface of the dimer is about 2100 Å2, which corresponds to about 15.3% of the accessible surface area of each molecule. There are 18 hydrogen bonds between the two molecules, and 62% of the atoms at the interface are non-polar atoms suggesting that the dimer is held together by both hydrophobic and polar interactions (Fig. 4B). In the case of Met276Ala structure, there are two molecules in the asymmetric unit, and the two are related by non-crystallographic 2-fold axis. Furthermore, the configuration of the dimer is the same as that in the wild type. Although the gel filtration results of both the wild type and the Met276Ala mutant suggest that TmFabK exists as a mixture of dimer and monomer in solution, the non-denaturing gel electrophoresis suggests that FabK is a dimer as shown in Fig. 4C. FabK is found as a dimer in both solution and crystal, therefore it is likely that FabK functions as a dimer. It is worth mentioning that SpFabK [8] and PgFabK [28] also form dimers with interface similar to that of Pa2ND and SaNAO [4,29] in the crystal form. 4. Conclusion The results of the structural and biochemical studies described here show that the hypothetical protein TM0800 gene product is a FabK with a preference of NADH. Mutagenesis studies show that FMN is also important for the activity since mutations on the residues involved in FMN abrogates the enzymatic activity of TmFabK. Conflict of interest The authors have no financial conflicts of interest. Acknowledgments We thank Dr. Kook Han Kim and the staff at beamline 5C of the Pohang Accelerator Laboratory, Korea and beamline AR-NW12 of the Photon Factory, Japan. This work was supported by grant from the Global Research Laboratory program of the Ministry of Science, ICT and Future Planning of Korea (NRF-2011-0021713), and support

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Please cite this article in press as: B.H. Ha, et al., Structural and biochemical characterization of FabK from Thermotoga maritima, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.141