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Biochemical and Structural Analysis of FomD that Catalyzes the Hydrolysis of Cytidylyl (S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis Shusuke Sato, Akimasa Miyanaga, Seung-Young Kim, Tomohisa Kuzuyama, Fumitaka Kudo, and Tadashi Eguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00690 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018
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Biochemistry
Biochemical and Structural Analysis of FomD that Catalyzes the Hydrolysis of Cytidylyl (S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis Shusuke Sato,† Akimasa Miyanaga,† Seung-Young Kim,‡,¶ Tomohisa Kuzuyama,‡,§ Fumitaka Kudo,*,† and Tadashi Eguchi*,† †
Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Biotechnology Research Center, §Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
‡
Supporting Information Placeholder
ABSTRACT: In fosfomycin biosynthesis, the hydrolysis of cytidylyl (S)-2-hydroxypropylphosphonate ((S)-HPPCMP) to afford (S)-HPP is the only uncharacterized step. Since FomD is an uncharacterized protein with a DUF402 domain that is encoded in the fosfomycin biosynthetic gene cluster, FomD was hypothesized to be responsible for this reaction. In the present study, FomD was found to hydrolyze (S)-HPP-CMP to give (S)-HPP and CMP efficiently in the presence of Mn2+ or Co2+. FomD also hydrolyzed cytidylyl 2-hydroxyethylphosphonate (HEPCMP), which is a biosynthetic intermediate before Cmethylation. The kcat/KM value of FomD with (S)-HPPCMP was 10-fold greater than that with HEP-CMP, suggesting that FomD hydrolyzes (S)-HPP-CMP rather than HEP-CMP in bacteria. The crystal structure of FomD showed that this protein adopts a barrel-like fold, which consists of a large twisted antiparallel β-sheet. This is a key structural feature of the DUF402 domain-containing proteins. Two metal cations are located between the FomD barrel and the two α-helices at the C-terminus, and serve to presumably activate the phosphonate group of substrates for hydrolysis. Docking simulations with (S)HPP-CMP suggested that the methyl group at the C2 position of the HPP moiety is recognized by a hydrophobic interaction with Trp68. Further mutational analysis suggested that a conserved Tyr107 among the DUF402 domain family of proteins activates a water molecule to promote nucleophilic attack on the phosphorus atom of the phosphonate moiety. These findings provide mechanistic insights into the FomD reaction and lead to a complete understanding of the fosfomycin biosynthetic pathway in Streptomyces.
Fosfomycin (Scheme 1) is a phosphonate-containing antibiotic that exhibits broad-spectrum antibacterial activity by inhibiting UDP-N-acetylglucosamine enolpyruvyltransferase, which catalyzes the condensation of phosphoenolpyruvate (PEP) with UDP-N-acetylglucosamine in the early stages of cell wall biosynthesis.1-4 Fosfomycin has a unique structure in which a phosphonate group is directly bonded to methyloxirane. Several species of Streptomyces and Pseudomonas produce fosfomycin, although the biosynthetic pathways are divergent.5-7 The first step of fosfomycin biosynthesis is the PEP phosphomutase reaction consisting of the cleavage of oxygen– phosphorus bond of PEP and the formation of carbon– phosphorus bond to afford phosphonopyruvate (PnPy) in both Streptomyces and Pseudomonas (Scheme 1).4 In Streptomyces, Fom1, which has a PEP phosphomutase domain at the C-terminus, is responsible for this reaction. PnPy is then converted to 2-hydroxyethylphosphonate (HEP) through decarboxylation by Fom2 and reduction by FomC. Subsequently, the N-terminal domain of Fom1 catalyzes the cytidylylation of HEP with CTP to afford cytidylyl 2-hydroxyethylphosphonate (HEP-CMP).8 Then, a cobalamin-dependent radical SAM C-methyltransferase, Fom3, catalyzes C-methylation at the C2 position of the HEP moiety of HEP-CMP to afford cytidylyl (S)-2-hydroxypropylphosphonate ((S)-HPP-CMP).9 The C-methylation catalyzed by Fom3 is (S)-selective and proceeds with inversion of the configuration.10,11 (S)HPP-CMP appears to be hydrolyzed to (S)-HPP by an uncharacterized hydrolase because (S)-HPP is converted to fosfomycin by non-heme iron-dependent peroxidase Fom4 during the final step of fosfomycin biosynthesis.12,13 The Fom4 catalyzed transformation is shared in both Streptomyces and Pseudomonas.
INTRODUCTION
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Scheme 1. The Fosfomycin Biosynthetic Pathway in Streptomyces O Fom1 O Phosphomutase domain HO P OH HO
HO P HO O
O Phosphoenolepyruvate (PEP)
O
HO P O
P
HO P O O
OH NH2
O
N
HO Fom3
O
O OH
Fom2
O
Phosphonopyruvate (PnPy)
O
N
O
HO
OH OH HEP-CMP
O
O
HO P H HO Phosphonoacetoaldehyde (PnAA)
Fom1 Cytidylyltransferase domain OH
FomC
HO P HO 2-Hydroxyethylphosphonate (HEP)
Fom4
HO P HO H
CH3
O P O O
OH NH2 N O
N
FomD
O
OH OH (S)-HPP-CMP
FomD is the only uncharacterized protein, which is encoded adjacent to the fom4 gene in the central fom gene cluster (Table S1).6 Because a fomD gene knockout mutant has never been obtained, the function of the fomD gene remains elusive.6 FomD is a representative of the DUF402 domain-containing proteins. In 2016, a DUF402 domain-containing protein SA1684 (13% identity and 35% similarity to FomD) was identified to catalyze the hydrolysis of nucleoside diphosphate in the presence of Mn2+ or Co2+ to give nucleoside monophosphate and inorganic monophosphate to enable the emergence of pathogenicity of Staphylococcus aureus.14 FomD also shows 31% identity to SC4828 from Streptomyces coelicolor, whose crystal structure has been deposited in the Protein Data Bank (PDB IDs: 3CBT, 3EXM). Although functional analysis of SC4828 has not been reported, the complex structure with the phosphomethylphosphonic acid guanosyl ester (PDB ID: 3EXM) suggests that SC4828 may catalyze the hydrolysis of a nucleoside phosphate. Therefore, we hypothesized that FomD is responsible for the hydrolysis of (S)-HPP-CMP to afford (S)-HPP. In the present study, we describe in vitro functional analysis of FomD and present the crystal structure of FomD to reveal the reaction mechanism of a DUF402-containing protein. MATERIALS AND METHODS Expression and Purification of SwFomD. The codon optimized fomD gene derived from Streptomyces wedmorensis (SwFomD, GenBank: BAA32492.1) for protein expression in Escherichia coli was obtained from Eurofins Genomics (Tokyo, Japan), which was designed to introduce a SpeI site, the thrombin recognition sequence and a HindIII site on the 3'-terminus as follows; 5'-CATfomD gene-ACTAGTCTGGTCCCTAGAGGCTCTAAGCTT-3'. The NdeI-HindIII fragment of the plasmid with the SwfomD gene was inserted into the same restriction enzyme sites of the expression vector pET30a (Novagen) to obtain the plasmid pET30-SwFomD. The obtained plasmid was introduced into E. coli BL21(DE3) cells for
O HO P HO
CH3 OH
(S)-2-Hydroxypropylphosphonate ((S)-HPP)
O CH3 H
O Fosfomycin
overexpression of SwFomD. E. coli harboring the pET30SwFomD were grown (200 rpm agitation) in 500 mL baffled flasks of LB medium (200 mL) containing 30 µg/mL kanamycin at 37 °C until the OD600 = 0.6–0.8. Protein expression was induced by the addition of isopropyl β-Dthiogalactopyranoside to a final concentration of 0.2 mM. Cultivation was continued at 15 ˚C for 20−24 h with 200 rpm agitation. The cells were harvested by centrifugation, washed with buffer A [50 mM HEPES-NaOH (pH 8.0), 10% (v/v) glycerol] and stored at −30 °C until use. The wet cells were suspended in buffer A. The suspension of cells were disrupted by sonication with sonication bursts of 5 sec with a 5 sec interval (total 2 min) at 4 °C (ice bath). Cell debris was removed by centrifugation. The supernatant was loaded onto a His60 Ni super flow resin (Clontech, Mountain View, CA) column (15ø × 15 mm) that had been pre-equilibrated with buffer A. The column was washed with 100 mL of buffer A containing 20 mM imidazole. SwFomD was eluted with buffer A containing 200 mM imidazole. The protein solution was collected and desalted with a PD-10 desalting column (GE Healthcare, Buckinghamshire, U.K.). The His-tagged SwFomD was concentrated with an Amicon Ultra 10K centrifugal filter (Merck Millipore) at 4,500 g and 4 °C and then used for functional assays. The concentration of SwFomD was determined by UV absorption at 280 nm using an extinction coefficient (ε280nm = 77.8 mM–1cm–1) determined by the Edelhoch’s method.15 Enzymes were stored at −80 °C until use. Expression and Purification of SfFomD. The fomD gene derived from Streptomyces fradiae (SfFomD, GenBank: ACG70828.1) was amplified by PCR with primers using the genomic DNA as a template DNA with the following primers, 5'-GGGGAATTCGCAGAAGTCGCCTCCCAGGA-3' and 5'-GGGAAGCTTCTAGGATCCTTCGGTCTCGA-3'. The EcoRI-HindIII fragment of the fomD gene was inserted into the same restriction sites of the expression vector pHis8,16 and then introduced into E. coli BL21(DE3) cells to yield a cell expression system. A PCR error causing the L139F mutation detected
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Biochemistry
by sequence analysis was repaired by site-directed mutagenesis. The site-directed mutagenesis was performed with the QuikChange method (Agilent Technologies, Santa Clara, CA) using the plasmid as a template with the following primers: 5'-GACTCGCGGACCCTGCGCTGGAAGGACG-3' and 5'-CGTCCTTCCAGCGCAGGGTCCGCGAGTC-3'. The plasmid was introduced into E. coli BL21(DE3) cells. The wild-type and L139F SfFomD were expressed and purified in the same manner as SwFomD. Although the wild-type protein was shown to aggregate by size-exclusion chromatography analysis, the L139F of SfFomD did not form aggregates under the purification conditions used. (Figure S1). The deposited sequence of the SfFomD may be incorrect, since our PCR amplification of the corresponding gene afforded the L139F variant with reproducibility. Thus, we describe the variant L139F as SfFomD from this point forward. The concentration of SfFomD was determined by UV absorption at 280 nm using an extinction coefficient (ε280nm = 76.5 mM–1cm–1) determined by the Edelhoch’s method.15 Enzymes were stored at –80 °C until required. 31
P-NMR Analysis for the Hydrolysis of (S)-HPP-CMP. HEP-CMP, (S)- and (R)-HPP-CMP were prepared according to previous reports.8,9 CTP, CDP and CMP were purchased from Sigma-Aldrich (St. Louis, MO). Five mM of (S)-HPP-CMP and 1 mM of MnCl2 were added to 4 µM of SwFomD and reacted at 28 °C for 10 min in buffer A. The addition of 10 mM of ethylenediaminetetraacetic acid (EDTA) quenched the reaction. The reaction solution was diluted 5-fold with D2O. The NMR data were recorded on a JEOL ECS-400 spectrometer (Tokyo, Japan). High Performance Liquid Chromatography (HPLC) Analysis of CMP Production. Ten microliters of the reaction solution was injected into an HPLC system [Senshu SSC-3215 degasser, Hitachi LaChrom ELITE pump L-2130, LaChrom ELITE Diode Array Detector L2455, Senshu SSC-3215 column oven] equipped with an InertSustain® AQ-C18 column (5 µm, 4.6 × 250 mm, GL Sciences Inc., Tokyo, Japan) equilibrated with 20 mM phosphate buffer (pH 2.3). The elution was made with a flow rate of 1.0 mL/min at 40 °C using an isocratic 20 mM phosphate buffer (pH 2.3). The elution was monitored at 280 nm. CMP was eluted at a retention time of 4.5 min. Standard curves were generated with authentic CMP so as to correlate peak area with the amount of CMP. Metal Requirement Analysis. Four micromolar SwFomD was incubated with 10 mM EDTA on ice for 1 h. The solution was loaded onto a PD-10 desalting column to remove metal ions present in the enzyme solution. Then, 5 nM of SwFomD and 100 µM of (S)-HPP-CMP were reacted in the presence of 50 µM of MgCl2, CaCl2, MnCl2, Fe(NH4)2(SO4)2, CoCl2, NiCl2, CuCl2, or ZnCl2 at 28 °C. After reacting for 1 min, 10 µL of a reaction solution was injected into the HPLC system.
Substrate Specificity Analysis. 100 µM of (S)- or (R)HPP-CMP, HEP-CMP, CTP, or CDP was added to a solution of 5 nM of SwFomD in the presence of 50 µM MnCl2 and reacted at 28 °C in buffer A. After reacting for 1 min, 10 µL of a reaction solution was injected into the HPLC system. FomD Reaction in H218O. The reaction solution was prepared with 1 µL of 560 µM SwFomD, 2.5 µL of 100 mM (S)-HPP-CMP, 0.2 µL of 50 mM MnCl2, 0.5 µL of 2 M HEPES-NaOH buffer (pH 8.0) and 95.8 µL of H218O (18O ≥ 97 atom%, Sigma-Aldrich). Consequently, the reaction solution contained 90 atom% of the 18O isotope. The reaction was carried out at 28 °C for 10 min and quenched by addition of EDTA. Ten microliters of the reaction solutions that were deproteinized with an Amicon Ultra device at 14,000 g and 4 °C were injected into a liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS) instrument (Shimadzu LCMS-2020 mass spectrometer equipped with LC-20AD pump, CTO20A column oven, FCV-20AH2 valve unit and SPDM20AUV detector). A TSK-GEL Amide-80 column (3 µm, 2.0 × 150 mm, TOSOH, Tokyo, Japan) pre-equilibrated in 85% CH3CN and 15% H2O containing 10 mM of ammonium formate (pH 3.2) was used for separation. The elution was made with a flow rate of 0.3 mL/min at 40 °C using ammonium formate buffer and CH3CN: 85% CH3CN for 10 min, 85–30% CH3CN over 10–20 min and 30% CH3CN for 20–25 min. (S)-HPP eluted as a broad peak and a retention time of 13.5–15 min, and CMP eluted at a retention time of 17 min. Kinetic Analysis. The assay solution contained 50 mM HEPES-NaOH buffer (pH 8.0), 10% (v/v) glycerol, 50 µM MnCl2, 0.63 nM SwFomD or SfFomD and 5–100 µM (S)-HPP-CMP. For (R)-HPP-CMP or HEP-CMP, the concentration of SwFomD and (R)-HPP-CMP or HEP-CMP were 4.5 nM and 10–150 µM, respectively. The reaction was initiated by the addition of substrate to the mixture and incubated at 28 °C. After reacting for 1 min, 10 µL of a reaction solution was injected into the HPLC system. The production of CMP was measured by HPLC and the value was taken as an initial velocity. Crystallization, Data Collection and Structure Determination. The His-tagged SwFomD and SfFomD were not crystallyzed at all. Thus, after the His-tag was cleaved by thrombin (GE Healthcare), SfFomD was further purified by size-exclusion chromatography. SfFomD (0.5 mg/mL) and 0.5 U/mL of thrombin were mixed and reacted overnight at 20 °C in buffer B [137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4, 10% (v/v) glycerol]. The reaction solution was loaded onto a His60 Ni superflow resin column, which had been pre-equilibrated with buffer B. The flow-through fraction was collected and concentrated with an Amicon concentrator. The solution of His-tag free SfFomD was loaded onto a Superdex 200 10/300 GL (GE Healthcare) column,
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which had been equilibrated with buffer C [20 mM HEPES−NaOH (pH 8.0), 150 mM NaCl, 10% (v/v) glycerol]. The protein was eluted with buffer C at a flow rate of 0.2 mL/min at 4 °C and desalted with a PD-10 column. The highly purified SfFomD was concentrated with an Amicon concentrator and used for crystallization screening. Crystals of ligand-free SfFomD were grown using sitting-drop vapor diffusion by mixing the protein solution [12.4 mg/mL in 20 mM HEPES-NaOH (pH 8.0) and 10% (v/v) glycerol] with an equal volume of reservoir solution [100 mM Tris-HCl (pH 8.5), 200 mM CaCl2, 30% (w/v) PEG 8000, 1% (v/v) trimethylamine N-oxide and 10 mM cytosine] at 5 °C. Crystals of SfFomD in complex with CDP were grown using sitting-drop vapor diffusion by mixing the protein solution [7.3 mg/mL in 20 mM HEPES-NaOH (pH 8.0) and 10% (v/v) glycerol] with an equal volume of reservoir solution [100 mM Tris-HCl (pH 8.5), 200 mM MgCl2, 30% (w/v) PEG 8000, 3% (v/v) ethanol and 10 mM CDP] at 5 °C. Prior to X-ray data collection, crystals were soaked in a cryoprotectant solution containing 25% (v/v) glycerol and flash-frozen in a liquid nitrogen stream. Diffraction data were collected at the AR-NW12A beamline of the Photon Factory (Tsukuba, Japan) and processed with the XDS software.17 The initial phase was determined by molecular replacement using the Molrep program18 with the phosphatase SC4828 structure (PDB ID: 3EXM) as the search model. The protein model building of the SfFomD was carried out automatically with the ARP/wARP program19 and subsequently inspected by Coot.20 Refmac21 was used to refine the structure. The structural representations were prepared with PyMOL (DeLano Scientific LLC, Palo Alto, CA). The geometries of the final structure were evaluated using the program MolProbity.22 The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB IDs: 5ZDM and 5ZDN). Docking Analysis. The docking study was carried out using the AutoDock 4.2 program.23 The (S)-HPP-CMP molecule was generated by using the PRODRG2 server.24 Chain A of the complex structure of SfFomD with CDP was used for the docking study. Although all water molecules and the CDP molecule of the FomD structure were removed for the docking study, two magnesium atoms that are coordinated to Asp129 were retained. Using AutoDockTools, polar hydrogen atoms were added to amino acid residues, and Gasteiger charges were assigned to all atoms of the protein. A charge was assigned to the two magnesium atoms. The torsion angles of (S)-HPP-CMP were rotatable. All of the protein residues were kept rigid. Grid maps were prepared with 40 × 40 × 40 points covering the substrate binding pocket with a point spacing of 0.375 Å. A total of 256 docking runs were performed by the Lamarckian genetic algorithm and the resulting binding modes were ranked into clusters based on their binding energies. The first-ranked cluster was reasonable large (98 of 256 docking runs).
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Mutational Analysis. Site-directed mutagenesis was performed with the QuikChange method (Agilent Technologies) using the plasmid pHis8-SffomD_L139F as a template with the following oligonucleotides and their complementary oligonucleotides: Y107F, 5'TCCGGGAGTGGTTCGTGAACGTGGAAGCG-3' and K142A, 5'-TCCGCTGGGCCGACGTGGAGAAGTTCGAG-3'. The variants of SfFomD were expressed and purified in the same manner as SwFomD. The assay solution contained 50 mM HEPES-NaOH buffer (pH 8.0), 10% (v/v) glycerol, 50 µM MnCl2, 300 nM SfFomD_Y107F and 2–80 µM (S)-HPP-CMP. For SfFomD_K142A, the concentration of SfFomD_K142A and (S)-HPP-CMP were 5 nM and 30–400 µM, respectively. Kinetic analysis was carried out using the same approach as the wild-type protein. RESULTS AND DISCUSSION Characterization of FomD. SwFomD derived from S. wedmorensis, which is one of the fosfomycin-producing strains,1 was heterologously expressed in E. coli and purified by Ni2+-affinity column chromatography (Figure S2). Size-exclusion chromatography showed that SwFomD exists as a monomer in solution (Figure S1). (S)HPP-CMP was reacted with SwFomD in the presence of Mn2+ as the divalent metal ion, and hydrolytic activity was analyzed by proton-decoupled 31P-NMR. Two doublet signals at 15 ppm and –11 ppm that represent (S)HPP-CMP decreased in intensity and two new signals appeared at 21 ppm and 4 ppm, corresponding to (S)-HPP and cytidine monophosphate (CMP), respectively (Figure 1). Thus, FomD catalyzes the hydrolysis of (S)-HPP-CMP. Next, we investigated the metal ion requirement of the FomD reaction. SwFomD and (S)-HPP-CMP were reacted in the presence of 50 µM Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, or Zn2+. HPLC analysis of CMP formation revealed that the reaction proceeds efficiently in the presence of Mn2+ or Co2+ (Figure S3). This enzymatic property is the same as that of SA1684, suggesting that hydrolases with the DUF402 domain commonly require Mn2+ or Co2+ for activity.
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Biochemistry
A (a)
a
(b) NH2
O -O P OH
b
[M-H]-
OH
O -O P O OH
139.07
N O
N
O
OH OH
[M-H]- 322.19
c
B (c)
(d)
d
e 20
12
4 [ ppm ]
-4
-12
Figure 1. 31P-NMR analysis of the FomD reaction (162 MHz, D2O). (a) Authentic sample of (S)-HPP-CMP, (b) authentic sample of CMP (c) authentic sample of (S)-HPP, (d) incubation of (S)-HPP-CMP with SwFomD, (e) incubation of (S)-HPP-CMP with boiled (denatured) SwFomD. Substrate Specificity of FomD. To investigate the substrate specificity of FomD, (S)-HPP-CMP, (R)-HPP-CMP, HEP-CMP, CDP and CTP were reacted with SwFomD. (R)-HPP-CMP and HEP-CMP were hydrolyzed with less efficiency than (S)-HPP-CMP (Figure S4), whereas CDP and CTP were not hydrolyzed by FomD. The kinetic constants of FomD using the Michaelis-Menten equation are summarized in Table 1. The KM and kcat values for hydrolysis of (S)-HPP-CMP by SwFomD were estimated to be 19 ± 2 µM and 75 ± 6 sec–1, respectively (Figure S5, Table 1). The KM value with HEP-CMP was approximately three to seven times higher than that with (S)-HPP-CMP, suggesting that the C2 methyl group of HPP-CMP is important for substrate recognition by FomD. The kcat/KM values with HEP-CMP dropped greatly under in vitro enzymatic reaction conditions, suggesting that FomD preferentially hydrolyzes (S)-HPP-CMP rather than HEPCMP in bacteria. FomD Reaction in H218O. The reaction of FomD was carried out in a buffer prepared with H218O to determine which phosphorus atom is attacked by a water molecule during hydrolysis. (S)-HPP-CMP was reacted with SwFomD in a H218O buffer at 28 °C for 10 min. LC-ESIMS analysis of the generated HPP in the negative mode showed m/z = 141, which is 2 Da larger than the species determined from the control reaction in distilled water, indicating that the 18O atom was incorporated into HPP (Figure 2). [M-H]– corresponding to CMP was unchanged at m/z = 322 (Figure 2). Therefore, it was found that a water molecule nucleophilically attacks the phosphorus atom of phosphonate between the two phosphorus atoms of (S)HPP-CMP in the FomD reaction.
125 130 135 140 145 305 310 315 320 325 m/z
Figure 2. LC-ESI-MS analysis of the FomD reaction in H218O. (A) MS spectrum of the FomD reaction in a buffer prepared with distilled water at a retention time of (a) 14 min (HPP) and (b) 17 min (CMP). (B) MS spectrum of the FomD reaction in a buffer prepared with H218O at a retention time of (c) 14 min (HPP) and (d) 17 min (CMP). All analyses were carried out in the negative mode. Crystal Structural Analysis of FomD. Since SwFomD was not crystallized despite several attempts, we used SfFomD which is derived from another fosfomycin producer strain S. fradiae1 (92 % identity to SwFomD) for crystallization. The kinetic constants of SfFomD showed similar values of KM = 9.4 ± 3.1 µM and kcat = 51 ± 4 sec– 1 (Figure S6, Table 1). The crystal structure of ligand-free SfFomD was solved at 1.38 Å resolution (Figure 3, Table S2). SfFomD comprises a large twisted antiparallel βsheet composed of eleven β-strands (β1–β11) with two additional α-helices located at the C-terminus. SfFomD contains two Ca2+ ions, which were presumably derived from the crystallization buffer. These two metal ions are coordinated by Asn109, Asp125, Glu127, Asp129 and Asp143 at the active site of FomD, and these residues are highly conserved among the FomD homologues and SA1684 (Figure 4B, C). Phe139 of SfFomD is on the surface of the protein, approximately 15 Å distal from the metal-binding site and does not appear to affect the catalytic activity of FomD. A search for structurally similar proteins using the DALI server25 revealed that SfFomD is structurally similar to two proteins with the DUF402 domain (Figure S7): SC4828 from S. coelicolor (PDB ID: 3CBT, Z-score 24.5, r.m.s.d. 2.2 Å) and the protein of unknown function DUF402 from Rhodococcus sp. PHA1 (PDB ID: 2P12, Z-score 12.1, r.m.s.d. 2.8 Å). The characteristic feature of their protein structures is annotated as the FomD barrel-like fold, which consists of a large twisted antiparallel β-sheet, in the SCOP database.26
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Table 1. Steady-State Kinetic Analysis of SwFomD and SfFomD kcat [sec–1]
KM [µM]
kcat/KM [sec–1µM–1]
Substrate
SwFomD
SfFomD
SwFomD
SfFomD
SwFomD
SfFomD
(S)-HPP-CMP
19 ± 2
9.4 ± 3.1
75 ± 6
51 ± 4
3.9
5.4
(R)-HPP-CMP
31 ± 5
21 ± 3
19 ± 1
10.8 ± 0.3
0.61
0.52
HEP-CMP
52 ± 10
68 ± 25
20 ± 2
14 ± 2
0.38
0.21
Arg74, Gln76 and Ser77 (Figure 4A). The structural positions of the three aromatic residues, especially Phe71, and the loop containing Arg74, Gln76 and Ser77 are slightly different from those of the ligand-free form to accommodate the pyrimidine ring of CDP (Figure S10).
Figure 3. Ribbon representation of the structure of ligandfree SfFomD. The Ca2+ ions are shown as pink spheres. Complex Structure with CDP. The complex structure of SfFomD with CDP was also solved at 2.02 Å resolution (Figure 4, Figure S8, Table S2). Because CDP inhibited the hydrolysis of (S)-HPP-CMP by FomD (Figure S9), CDP was expected to dock within the active site of FomD. In fact, CDP bound to FomD with little overall structural perturbation to the SfFomD polypeptide backbone (r.m.s.d. of 0.69 Å for superposition of the Cα atoms). The complex structure with CDP contains four Mg2+ ions, which were presumably derived from the crystallization buffer (Figure 5A, Figure S8). Two Mg2+ ions are coordinated at the same positions as the metal ions in the ligandfree form and additional two Mg2+ ions are coordinated to the phosphate group of CDP. The two phosphate groups of CDP are exposed to the solvent and are not in close proximity to the two metal ions at the active site of FomD, which might explain why CDP was not hydrolyzed by FomD. The pyrimidine ring of CDP is located within the hydrophobic pocket formed by the three aromatic residues, Trp68, Phe71, Trp75, which are highly conserved among FomD family members (Figure 4A, C). The pyrimidine ring also forms hydrogen bonds with the main chain of
Docking Simulation with (S)-HPP-CMP. The complex structure with CDP did not provide significant insight into the hydrolytic mechanism by FomD, because the position of pyrophosphate moiety of CDP deviates from the active site. Although we attempted to cocrystallize SfFomD with (S)-HPP-CMP, we could not solve the complex structure with (S)-HPP-CMP. Therefore, we conducted docking simulation analysis with (S)-HPP-CMP using the AutoDock4.2 program.22 In the obtained docking model, the pyrimidine moiety of (S)-HPP-CMP is bound to the protein in a similar manner to that observed for the CDPFomD complex; although, the position of the phosphate is different (Figure 5). The phosphonate moiety of (S)-HPPCMP is bridged between two metal ions in the docking model (Figure 5B). One metal is coordinated by both the 2-OH group and phosphonate group of (S)-HPP-CMP. The model structure indicated that the methyl group at the C2 position of the HPP moiety is recognized by a hydrophobic interaction with Trp68 (Figure 5B). HEP-CMP could not interact with Trp68 because this substrate does not have the methyl group at the C2 position, which might explain why the activity of FomD with HEP-CMP was lower than that with (S)-HPP-CMP. Since Trp68 is also involved in the recognition of the pyrimidine ring, it appears to be a crucial residue in substrate recognition by FomD. The phosphate group of (S)-HPP-CMP is hydrogen bonded to Lys142 in the docking model (Figure 5B). Tyr107, which does not coordinate to metal ions, is also located close to the phosphonate group in the docking model. Therefore, Tyr107 and Lys142 are likely to be involved in the activation of a water molecule in the FomD catalytic cycle. Multiple alignment analysis of FomD homologues shows that Tyr107 and Lys142 are conserved among the homologues and SA1684, suggesting that these amino acids are crucial for the enzymatic activity of this family of enzymes (Figure 4C).
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Biochemistry
A
B K142
3.5
S77
Y107
D129 D143
2.7
N109 3.0
W75
W68
2.0
2.0 2.1
F71
2.1
2.0 2.2
2.1 1.9
E127
D125 2.9
W68
C
Figure 4. (A) The binding site of the pyrimidine moiety of CDP. Hydrogen bonds are shown as yellow dashed lines. All distances are shown in Å. (B) The binding site of the two metal ions. Coordinate bonds are shown as yellow dashed lines. The metal ions are shown as pink spheres and a water molecule is shown as a cyan sphere. (C) The alignment of SfFomD with the eight homologues and SA1684. FomD from S. wedmorensis (identity 91%), DUF402 domain-containing protein from S. vidochrimogenes (identity 68%), DUF402 domain-containing protein from Kitasatospora purpeofusca (identity 54%), hypothetical protein UK_07560 from Saccarothrix sp. ST-888 (identity 61%), hypothetical protein ACZ90_14195 from S. albus subsp. albus (identity 50%), DUF402 domain-containing protein from Nocardia beijingensis (identity 41%), hypothetical protein ADL17_10000 from Verrucosispora maris (identity 43%) and SC4828 from S. coelicolor (identity 31%). The amino acid sequences were aligned by ClustalW (http://clustalw.ddbj.nig.ac.jp) and presented using the ESPript 3.0 (http://sprint.ibcp.fr/ESPript/ESPript/) program. The FomD residues that interact with the pyrimidine ring of CDP are indicated with orange circles. The FomD residues that are involved in the metal coordination are indicated with green circles. Tyr107 and Lys142 of SfFomD are indicated with red circles.
Mutational Analysis. Mutational analysis was conducted to clarify the roles of Tyr107 and Lys142 of FomD. Tyr107 and Lys142 of SfFomD were mutated to phenylalanine and alanine, respectively. The KM value of Y107F was similar to that of the wild-type protein, whereas the
kcat value was approximately 250-fold lower (Figure S11), indicating that Tyr107 is a catalytic residue. The KM value of K142A increased approximately 10-fold (Figure S12) when compared with that of the wild-type protein. Therefore, Lys142 appears to be involved mainly in substrate recognition.
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Proposed Hydrolytic Mechanism by FomD. A plausible hydrolytic mechanism of (S)-HPP-CMP catalyzed by FomD is shown in Figure 6. Initially, (S)-HPP-CMP coordinates two divalent metal ions through the phosphonate group. Tyr107 activates a water molecule as a base to promote nucleophilic attack on the phosphorus atom of the phosphonate group. The metal ion activates the phosphonate as a Lewis acid, and then the P–O–P bond in (S)HPP-CMP is cleaved to give (S)-HPP and CMP. Lys142, which recognizes the phosphate group, would act as a proton source.
A
Y107
W68
B
Y107
W68 K142
K142
Figure 5. (A) The structure of active site in the complex with CDP. The CDP molecule is shown in cyan sticks. (B) The structure of active site in the docking model with (S)HPP-CMP. The docked (S)-HPP-CMP molecule is shown in yellow sticks. Coordinate bonds and hydrogen bonds are shown as yellow dashed lines and the metal ions are shown as magenta spheres. The extra Mg2+ ions are shown as light green spheres. Comparison with Binuclear Metallohydrolases. The characterization and structural analysis of FomD revealed that a hydrolase with a DUF402 domain would belong to a binuclear metallohydrolase. Binuclear metallohydrolases exist widely as hydrolases of amide bonds and carboxylate and phosphate esters.27,28 Although the metal ions needed for activity vary depending on the enzyme, it is common that the hydrolysis proceeds by coordinating the substrate to the metal ions. In the initial step of the reaction catalyzed by binuclear metallohydrolases, including inositol monophosphatase29,30 and fructose-1,6bisphosphatase,31,32 the carboxylate of the glutamic acid residue abstracts a proton from water to promote its
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nucleophilic attack to the phosphate ester. Interestingly, there is no such acidic amino acid at the active site of FomD. Instead, a highly conserved Tyr residue among the DUF402 domain-containing proteins including SA1684,14 appears to play the role of base. A 3'-5' exonuclease of E. coli DNA polymerase I also uses a Tyr residue for facilitating hydrolysis.33,34 The Role of the CMP Moiety in Fosfomycin Biosynthesis. In fosfomycin biosynthesis by Streptomyces, the Nterminal cytidylyltransferase domain in Fom1 is responsible for the attachment of the CMP moiety on HEP-CMP (Scheme 1). Although (S)-HPP is also cytidylylated by Fom1, the catalytic efficiency with (S)-HPP was 10-fold lower than that with HEP (Figures S12–13). The crystal structure of the cytidylyltransferase domain of Fom1 with HEP-CMP (PDB ID: 5X3D)8 showed that the methylene at the C2 position of the HEP moiety is arranged in a narrow pocket constituted by Leu37, Tyr45, Leu75, Tyr77 and Gln100, making it difficult for Fom1 to recognize (S)HPP (Figure S14), whereas FomD selectively recognizes (S)-HPP-CMP as mentioned above. Therefore, cytidylylation and hydrolysis of cytidylylate in fosfomycin biosynthesis appear to be well controlled by the substrate specificity of cytidylyltransferase Fom1 and hydrolase FomD. The CMP moiety of HEP-CMP is necessary for the substrate recognition and the stereoselective C-methylation by a radical SAM C-methyltransferase Fom3 to afford (S)-HPP-CMP, which is hydrolyzed by FomD. The generated (S)-HPP is then oxidized by Fom4 to give fosfomycin. The value of kcat/KM of Fom4 was estimated to be at least 120 mM–1sec–1 at 4 °C in the presence of H2O2.35 Overall, the attachment of CMP seems to be an intentional step to achieve the selective biosynthesis of fosfomycin. In conclusion, FomD, which had been annotated as an unknown functional protein in the fosfomycin biosynthetic gene cluster, was found to hydrolyze (S)-HPP-CMP to give (S)-HPP and CMP in the presence of Mn2+ or Co2+. The in vitro enzymatic analysis combined with structural data of FomD has enabled us to finalize the fosfomycin biosynthetic pathway that has been discussed for 50 years since the isolation and structure determination of the antibiotic fosfomycin.
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Biochemistry
Y107
Y107 M2+
M2+ (S)-HPP-CMP
M2+
OH
O-
P H 2O
NH W68
O
NH K142
H 3N
M2+
O
-O
O
-O
cytidine
H HO
P
O
W68
-O
H 3N
K142
H 2O
CMP (S)-HPP
Y107
Y107 M2+
M2+ OH
M2+
O O-
P
OH HO NH
O
W68 O cytidine
P
H 2N
O-
P
OH
NH
K142
O O
W68 cytidine
M2+
O-
HO
OH P
-O
O
H 3N
K142
OH
O-
Figure 6. Proposed mechanism of hydrolysis catalyzed by FomD.
Notes
ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publications website.
While this paper was in reviewing process, the enzymatic function of the FomD protein was reported.11 The authors declare no competing financial interest.
Supporting figures and tables
ACKNOWLEDGMENTS
AUTHOR INFORMATION
This work was performed with the approval of the Photon Factory Program Advisory Committee (Proposal 2016G624). This work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 18H02095, Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research in Innovative Areas (16H06451 to T.E. and 16H06453 to T.K.), and the Japan Society for the Promotion of Science A3 Foresight Program.
Corresponding Author
*
[email protected], *
[email protected] ORCID
Akimasa Miyanaga: 0000-0003-2219-6051 Tomohisa Kuzuyama: 0000-0002-7221-5858 Fumitaka Kudo: 0000-0002-4788-0063 Tadashi Eguchi: 0000-0002-7830-7104
ABBREVIATIONS
Present Addresses ¶
Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Chungnam 31460, Republic of Korea
Author Contributions
SS, AM, TK, FK, and TE designed the research; SS, AM and SYK performed the experiments; SS, AM, FK and TE analyzed data and wrote the manuscript; all authors approved the final version of the manuscript.
CTP, cytidine 5'-triphosphate; CDP, cytidine 5'-diphosphate; CMP, cytidine 5'-monophosphate; (S)-HPP, (S)-2hydroxypropylphosphonate; (S)-HPP-CMP, cytidylylated (S)-2-hydroxypropylphosphonate; HEP, 2-hydroxyethylphosphonate; HEP-CMP, cytidylylated, 2-hydroxyethylphosphonate; PEP, phosphoenolpyruvate; PnPy, phosphonopyruvate; EDTA, ethylenediaminetetraacetic acid; HPLC, high performance liquid chromatography; LCESI-MS, liquid chromatography and electrospray ionization mass spectrometry REFERENCES
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(1) Hendlin, D.; Stapley, E. O.; Jackson, M.; Wallick, H.; Miller, A. K.; Wolf, F. J.; Miller, T. W.; Chaiet, L.; Kahan, F. M.; Foltz, E. L.; Woodruff, H. B.; Mata, J. M.; Hernandez, S.; Mochales, S. (1969) Phosphonomycin, a New Antibiotic Produced by Strains of Streptomyces. Science 166, 122-123. (2) Christensen, B. G.; Leanza, W. J.; Beattie, T. R.; Patchett, A. A.; Arison, B. H.; Ormond, R. E.; Kuehl, F. A. Jr.; Albers-Schonberg, G.; Jardetzky, O. (1969) Phosphonomycin: Structure and Synthesis. Science 166, 123-125. (3) Shoji, J.; Kato, T.; Hinoo, H.; Hattori, T.; Hirooka, K.; Matsumoto, K.; Tanimoto, T.; Kondo, E. (1986) Production of Fosfomycin (Phosphonomycin) by Psedomonas syringae. J. Antibiot. 39, 1011-1012. (4) Horsman, G. P., Zechel, D. L. (2017) Phosphonate Biochemistry. Chem. Rev. 117, 5704-5783. (5) Hidaka, T., Goda, M., Kuzuyama, T., Takei, N., Hidaka, M., Seto, H. (1995) Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis. Mol. Gen. Genet. 249, 274-280. (6) Woodyer, R. D., Shao, Z., Thomas, P. M., Kelleher, N. L., Blodgett, J. A., Metcalf, W. W., van der Donk, W. A., Zhao, H. (2006) Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster. Chem. Biol. 13, 1171-1182. (7) Kim, S.-Y.; Ju, K.-S.; Metcalf, W. W.; Evans, B. S.; Kuzuyama, T.; van der Donk, W. A. (2012) Different biosynthetic pathways to fosfomycin in Pseudomonas syringae and Streptomyces Species. Antimicrob. Agents Chemother. 56, 4175-4183. (8) Cho, S.-H.; Kim, S.-Y.; Tomita, T.; Shiraishi, T.; Park, J. S.; Sato, S.; Kudo, F.; Eguchi, T.; Funa, N.; Nishiyama, M.; Kuzuyama, T. (2017) Fosfomycin biosynthesis via transient cytidylylation of 2-hydroxyethylphosphonate by the bifunctional Fom1 enzyme. ACS Chem. Biol. 121, 2209-2215. (9) Sato, S.; Kudo, F.; Kim, S.-Y.; Kuzuyama, T.; Eguchi, T. (2017) Methylcobalamin-dependent radical SAM C-methyltransferase Fom3 recognizes cytidylyl-2-hydroxyethylphosphonate and catalyzes the nonstereoselective C-methylation in fosfomycin biosynthesis. Biochemistry 56, 3519-3522. (10) Sato, S.; Kudo, F.; Kuzuyama, T.; Hammerschmidt, F.; Eguchi, T. (2018) C-Methylation catalyzed by Fom3, a cobalamin-dependent radical SAM enzyme in fosfomycin biosynthesis, proceeds with inversion of configuration. Biochemistry DOI: 10.1021/acs.biochem.8b00614 (11) McLaughlin, M. I.; van der Donk, W. A. (2018) Stereospecific radical-mediated B12-dependent methyl transfer by the fosfomycin biosynthesis enzyme Fom3. Biochemistry DOI: 10.1021/acs.biochem.8b00616 (12) Liu, P.; Murakami, K.; Seki, T.; He, X.; Yeung, S.-M.; Kuzuyama, T.; Seto, H.; Liu, H.-w. (2001) Protein Purification and function assignment of the epxidase catalyzing the formation of fosfomycin. J. Am. Chem. Soc. 123, 4619-4620. (13) Zhao, Z.; Liu, P.; Murakami, K.; Kuzuyama, T.; Seto, H.; Liu, H.w. (2002) Mechanistic studies of HPP epoxidase: configuration of the substrate governs its enzymatic fate. Angew. Chem. Int. Ed. Engl. 41, 4529-4532. (14) Imae, K.; Saito, Y.; Kizaki, H.; Ryuno, H.; Mao, H.; Miyashita, A.; Suzuki, Y.; Sekimizu, K.; Kaito, C. (2016) Novel nucleoside diphosphate contributes to Staphylococcus aureus virulence. J. Biol. Chem. 291, 18608-18619. (15) Edelhoch, H.(1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954. (16) Jez, J.; Ferrer, J.; Bowman, M.; Dixon, R.; Noel, J. (2000) Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39, 890-902.
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(17) Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132. (18) Vagin, A.; Teplyakov, A. (2010) Molecilar replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22-25. (19) Morris, R. J.; Perrakis, A.; Lamzin, V. S. (2002) ARP/wARP and automatic interpretation of protein electron density maps. Acta Crystallogr. D Biol. Crystallogr. 58, 968-975. (20) Emsley, P.; Cowtan, K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132. (21) Murshudow, G. N.; Vagin, A. A.; Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240-255. (22) Chen, V. B.; Arendall, W. B.; 3rd, Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Rhichardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12-21. (23) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785-2791. (24) Schüttelkopf, A. W; van Aalten, D. M. F. (2004) PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355-1363. (25) Holm, L.; Sander, C. (1998) Touring protein fold space with Dali/FSSP. Nucleic. Acids. Res. 26, 316-319. (26) Murzin, A. G.; Brenner, S. E.; Hunbbard, T.; Chothia, C. (1995) SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536-540. (27) Wilcox, D. E. (1996) Binuclear metallohydrolases. Chem. Rev. 96, 2435-2458. (28) Mitić, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.; Schenk, G. (2006) The catalytic mechanisms of binuclear metallohydrolases. Chem. Rev. 106, 3338-3363. (29) Bone, R.; Frank, L.; Springer, J. P.; Pollack, S. J.; Osborne, S. A.; Atack, J. R.; Knowles, M. R.; McAllister, G.; Ragan, C. I.; Broughton, H. B.; Baker, R.; Fletcher, S. R. (1994) Structural analysis of inositol monophosphatase complex with substrates. Biochemistry 33, 94609467. (30) Pollack, S. J.; Knowles, M. R.; Atack, J. R.; Broughton, H. B.; Ragan, C. I.; Osborne, S.; McAllister, G. (1993) Probing the role of the metal ions in the mechanism of inositol monophosphatase by site-directed mutagenesis. Eur. J. Biochem. 217, 281-287. (31) Zhang, Y.; Liang, J.-Y.; Huang, S.; Ke, H.; Lipscomb, W. N. (1993) Crystallographic studies of the catalytic mechanism of the neutral form of fructose-1,6-bisphosphatase. Biochemistry 32, 1844-1857. (32) Chen, L.; Hegde, R.; Chen, M.; Fromm, H. J. (1993) Site-specific mutagenesis of the metal binding sites of porcine fructose-1,6-bisphosphatase. Arch. Biochem. Biophys. 307, 350-354. (33) Derbyshire, V.; Grindley, N. D.; Joyce, C. M. (1991) The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 10, 17-24. (34) Brautigam, C. A.; Steitz, T. A. (1998) Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J. Mol. Biol. 277, 363-377. (35) Wang, C.; Chang, W.-c.; Guo, Y.; Huang, H.; Peck, S. C.; Pandelia, M. E.; Lin, G.-m.; Liu, H.-w.; Krebs, C.; Bollinger, J. M. Jr. (2013) Evidence that the fosfomycin-producing epoxidase, HppE, is a nonheme-iron peroxidase. Science 342, 991-995.
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Biochemistry
Table of Contents O O
Fom1
HO P OH CTP HO 2-Hydroxyethylphosphonate
HO P O HO P
Y107
OH NH2 N O
O
O
N
O HO P HO H
W68
O
OH OH Fom3 O HO HO
P
CH3 (S) OH
O P O O
FomD
N O
N
OH OH
Fom4
DUF402-containing protein
NH2 O
O HO P HO
Mn2+ or Co2+ H 2O
CMP
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CH3
H O Fosfomycin
K142
CH3 (S)
OH
