Crystal structure of methylesterase family member 16 (MES16) from Arabidopsis thaliana

Biochemical and Biophysical Research Communications 474 (2016) 226e231

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Crystal structure of methylesterase family member 16 (MES16) from Arabidopsis thaliana Hongmei Li a, *, Hua Pu a, b a

Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China b University of Chinese Academy of Sciences, College of Life Science, Beijing, 101048, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2016 Accepted 20 April 2016 Available online 22 April 2016

Methylesterase family member 16 (MES16) is an integral component of chlorophyll breakdown. It catalyzes the demethylation of fluorescent chlorophyll catabolite (FCC) and pheophorbide in vitro, and specifically demethylates FCC in vivo. Here we report the crystal structure of MES16 from Arabidopsis thaliana at 2.8 Å resolution. The structure confirm that MES16 is a member of the a/b-hydrolase superfamily with Ser-87, His-239, and Asp-211 as the catalytic triad. Our biochemical studies reveal that MES16 has esterase activity with methyl-indole acetic acid as the substrate, and the catalytically essential role of Ser-87 has been demonstrated. © 2016 Elsevier Inc. All rights reserved.

Keywords: Arabidopsis thaliana Chlorophyll breakdown MES16 Demethylation a/b-hydrolase superfamily Catalytic triad

1. Introduction Chlorophyll (Chl) breakdown is an important catabolic process of leaf senescence and fruit ripening. The reactions can be divided into two phases that are common to high plants. The early reactions take place within senescing chloroplasts and result in the formation of a colorless primary fluorescent Chl catabolite (pFCC) [1e3]. In later steps of the pathway of Chl breakdown, pFCC undergoes species-specific modifications at different peripheral positions to give rise to modified FCCs and hypermodified FCCs [4e7]. These reactions occur in the cytosol, implying that pFCC is exported from the chloroplast. The modified FCCs are then imported into the vacuole, where non-enzymatic isomerization of modified FCCs to their respective nonfluorescent Chl catabolites (NCCs) occur spontaneously because of the acidic pH of the vacuolar sap [8e10]. Besides these main steps aforementioned, additional reactions leading to other degradation products such as Chl-derived monopyrroles and different pigments with an intact porphyrin

Abbreviations: FCC, Fluorescent chlorophyll catabolite; Chl, Chlorophyll; pFCC, Primary fluorescent Chl catabolite; NCC, Nonfluorescent Chl catabolites; Pheide, Pheophorbide; PPD, Pheophorbidase; MeIAA, Methyl-indole acetic acid; MeJA, Methyl-jasmonic acid. * Corresponding author. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.bbrc.2016.04.115 0006-291X/© 2016 Elsevier Inc. All rights reserved.

ring have been described [11e14]. Among the latter, pyro (¼C132decarboxymethylated) forms of pheophorbide (Pheide) and pheophytin have been discussed as breakdown products of Chl [13,14]. Pheophorbidase (PPD) from radish (Raphanus sativus [RsPPD]) has been demonstrated to catalyze O134-demethylation of Pheide in vitro, however, this demethylation is followed by a spontaneous decarboxylation resulting in pyro-Pheide [15]. The chemical structures of NCCs from Arabidopsis indicate the presence of an enzyme activity that demethylates the C132-carboxymethyl group present at the isocyclic ring of Chl. Arabidopsis methylesterase16 (MES16), the protein most closely related to RsPPD, catalyzes the same reaction. Furthermore, in senescent leaves, MES16 was identified as the enzyme that hydrolyzes the methyl ester group of pFCCs. MES16-deficient mutants (mes16) were still able to degrade Chl, but FCC-NCC isomerization was compromised and the mutants accumulated large quantities of FCCs. MES16 protein was able to demethylate both Pheide and pFCC in vitro and specifically acts on FCCs in vivo [16]. MES16 has also been shown to hydrolyze two different methylated plant hormones, methyl-indole acetic acid (MeIAA) and methyl-jasmonic acid (MeJA), in vitro [17]. The competitive experiment for esterase activity showed that MES16 has a high preference for MeIAA as substrate in addition to Pheide a [16]. To help understanding of the biochemical and biological functions of MES16, we determined its 3D structure at up to 2.8 Å

H. Li, H. Pu / Biochemical and Biophysical Research Communications 474 (2016) 226e231

resolution. The structure confirms that MES16 is a member of the a/ b-hydrolase superfamily with Ser-87, His-239, and Asp-211 as the catalytic triad. Our biochemical studies reveal that MES16 has esterase activity with methyl-indole acetic acid (MeIAA) as the substrate, and the catalytically essential role of Ser-87 has been demonstrated. This work provides a structural insight into the function of MES16.

2. Materials and methods 2.1. Cloning, protein expression and purification Arabidopsis thaliana methylesterase16 gene (GenBank accession no. AEE83784.1; At4g16690) was cloned into pET-28a (Novagen) to generate the pET-28a (þ)-MES16 construct. This construct was verified by standard DNA sequencing analysis and then was transformed into expression strain Escherichia coli BL21 (DE3) cells. A single isolate was cultured in LB medium containing 0.1% kanamycin. Initial growth was carried out at 37  C until mid-log phase (OD600 ¼ 0.8), the temperature was then decreased to 18  C, and protein expression was induced by the addition of isopropyl-b-Dthiogalactopyranoside (IPTG) at a final concentration of 1 mM. After overnight incubation at 18  C, the cells were harvested by centrifugation. MES16 protein samples were purified using standard protocols. Briefly, cell pellets were resuspended in lysis buffer (20 mM TriseHCl, pH 7.5, 200 mM NaCl, 20 mM imidazole) and disrupted by sonication. The cell debris was removed via centrifugation at 39,000  g for 30 min. The supernatant was loaded onto a Ni-NTA column (QIAGEN) and eluted with lysis buffer containing increasing concentrations of imidazole (20, 50, 100 and 200 mM). Fractions containing the purified MES16 were pooled, concentrated by ultrafiltration, and loaded onto a gel filtration column (Superdex 200, 16/60, GE Healthcare) and eluted with buffer of 20 mM TriseHCl, pH 7.5, and 200 mM NaCl. The resulting purified MES16 protein was concentrated to 16 mg/ml, shock-frozen in liquid nitrogen, and stored at 80  C.

2.2. Site-directed mutagenesis MES16 mutant MES16 Ser87Ala was generated with Fast Mutagenesis System Kit (TransGen Biotech, Beijing) using pET-28a (þ)-MES16 as template. The MES16 Ser87Ala plasmid was sequenced to confirm the desired mutation. The procedure for purification of the MES16 Ser87Ala mutant protein was the same as that of the wild type.

2.3. Protein crystallization C

MES16 was crystallized at 4 using the sitting-drop vapor diffusion method by mixing 1 ml protein sample with an equal volume of crystallization solution in a 200 ml reservoir. Initial crystallization trials were performed using the commercial crystallization reagent kits Crystal Screen (Hampton Research) and Nextal Classic (QIAGEN) at 277 K and 289 K. Further optimization was conducted for conditions yielding crystals by variation of protein concentration, precipitant gradient, the pH, and by the use of additives (Hampton Research). Finally, crystals suitable for data collection were obtained at 289 K from the following improved condition: 100 mM sodium acetate trihydrate, pH 4.8, 10% (w/v) PEG 4000 and 20% (v/v) isopropanol. The crystals were transferred to 20% glycerol for cryo-protection, and then flash-frozen in liquid nitrogen for data collection.

227

2.4. Data collection and structure determination Diffraction data were collected at a wavelength of 0.9792 Å at 100 K on beamline BL-17U at the Shanghai Synchrotron Radiation Facility (SSRF) equipped with ADSC Quantum 315r detector. 20% glycerol was used as a cryoprotectant for data collection under cryogenic conditions. The diffraction data were indexed, integrated and scaled with DENZO and SCALEPACK as implemented in HKL2000 [18]. Molecular replacement was carried out with Phaser in the CCP4 suite [19,20]. Structure refinements were performed with phenix.refine and the overall quality of the final structural models was assessed by PROCHECK [21,22]. Data-collection and structurerefinement statistics are summarized in Table 1. The protein structure figures were prepared using PyMOL. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://www.pdb.org) under the accession code 5HK8.

2.5. Esterase enzyme assay Esterase assay of MES16 with MeIAA as substrate was performed using the coupled methyltransferase assay as previously described [17]. All experiments were performed in triplicate. Firstly, incubation of recombinant MES16 protein and MeIAA for 30 min in the reaction buffer (20 mM TriseHCl, pH 7.5, 200 mM NaCl) after which the enzyme was inactivated by boiling in 95  C. Then, the amount of indole-3-acetic acid (IAA) generated from the esterase assay was quantified by HPLC analysis on a Waters 2690 Separations Module. HPLC separation of MeIAA and IAA was achieved over a Waters Alltima C18 column, using an 8-min linear gradient from 65%

Table 1 X-ray Diffraction Data collection and Refinement Statictics. Data collection

MES16

Space group Wavelength (Å) Unit cell dimensions a, b, c (Å) a, b, g ( ) Resolution (Å)a Total/unique reflections Completeness (%) Redundancy I/s Rmergeb

C2221 0.9792 149.6, 176.4, 168.4 90.0, 90.0, 90.0 50.00e2.80 (2.90e2.80) 806841/54972 100 (100) 14.7 (15.1) 45.7 (4.1) 0.065 (0.676)

Refinement Resolution (Å) No. of reflections (work) No. of unique reflections (test) Rworkc/Rfreed No. of none-H atoms/average B-factor (Å) Protein Water Wilson B- factor (Å) R.m.s deviations Bond lengths (Å) Bond angles ( ) Ramachandran plot Favored (%) Allowed (%) Outliers (%) a

48.00e2.80 54904 2788 0.21/0.25 11167/59.60 217/42.82 73.09 0.005 0.910 96.19 3.81 0

The values in parentheses relate to the highest resolution shell. Rmerge ¼ Sh Si jI(h)i ej/Sh Si I(h)i, where I(h) is the intensity of reflection h, Sh is the sum over all reflections, and Si is the sum over i measurements of reflection h. c Rwork ¼ SjjFoj  jFcjj/SjFoj, where Fo and Fc are the observed and calculated structure factors, respectively. d Rfree is the cross-validated R-factor computed for a test set of 5% of the reflections, which were omitted during refinement. b

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H. Li, H. Pu / Biochemical and Biophysical Research Communications 474 (2016) 226e231

Fig. 1. Overall structure of MES16. (A) Topology diagram of the secondary structural elements. Helices and b-strands are represented by cylinders and arrows. The cap domain (orange) is inserted between b4 and b7. The dotted lines and cylinders indicate the missing region. Red dots denote the catalytic triad. (B) Ribbon representation of MES16 tetramer. The four monomers are colored in green, cyan, magenta, and yellow, and labeled with chain A, B, C and D respectively. (C) Ribbon representation of MES16 monomer. Side chains of residues forming the catalytic triad are shown in stick representation. Detection of chlorophyll breakdown products in the senescent leaves of higher plants.

Table 2 Structural homologs of MES16 identified by DALI. Protein name

Source

PDB code

Za

RMSDb (Å)

Lalic (n)

Identityd (%)

SABP2 MKS1 PNAE AtHNL BmHNL

Nicotiana tabacum Lycopersicon hirsutum Rauvolfia serpentina Arabidopsis thaliana Baliospermum montanum

1XKL 3STT 2WFM 3DQZ 3WWP

33.9 33.9 33.8 33.6 33.5

2.0 1.6 1.8 1.7 1.6

257 236 232 231 231

34 33 32 33 31

a b c d

Z score, strength of structural similarity in standard deviation above expected. Root mean square deviation of superimposed Ca atoms. Lali, number of equivalent residues. Percentage of sequence identity over equivalent position.

acetonitrile in 1.5% phosphoric acid to 90% acetonitrile, with the flow rate set at 1 ml/min and the column temperature set to 30  C. In-line UV light spectra (200e450 nm) were obtained using an attached Waters 996 photodiode array detector. Eluting compounds were identified by comparison of both UV light spectra and elution volume with authentic MeIAA and IAA. 3. Results 3.1. Structure determination of MES16 The crystal structure of Arabidopsis MES16 was determined at

2.8 Å resolution by molecular replacement using the structure of SABP2 from Nicotiana tabacum (PDB entry 1XKL) as search model. The crystals belong to space group C2221, with cell parameters of a ¼ 149.64 Å, b ¼ 176.35 Å, c ¼ 168.45 Å, and a ¼ 90 , b ¼ 90 , g ¼ 90 . The data-collection and refinement statistics are summarized in Table 1. 96.19% of the modeled residues lie in the most favored regions of the Ramachandran plot with no outliers. The final model has been refined to Rwork/Rfree values of 0.21/0.25 for 48.00e2.80 Å data.

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Fig. 2. Sequence alignment of Arabidopsis MES16 with SABP2 from N. tabacum, MKS1 from S. habrochaites, PNAE from R. serpentina, S-selective HNL from A. thaliana (AtHNL), and Rselective HNL from B. montanum (BmHNL). Identical amino acids are in white on a red background. The similar residues are in red and boxed in blue. Amino acids missed in the MES16 structure are boxed in red. The cap domains are boxed in black. The three catalytic triad residues are marked with red triangles.

3.2. Overall structure of MES16 The crystal structure confirms that MES16 is a member of the a/ b hydrolase superfamily. It generally adopts the canonical a/b-hydrolase fold (Fig. 1A). There are six monomers in an asymmetric unit, which are designated chains A-F. Chains A, B, C, and D form a tetramer (Fig. 1B) and chains E and F form a neighboring dimer (Fig. S1). The six monomers in the asymmetric unit are similar to

each other, with an average root mean square deviation (r.m.s.d.) of 0.21 Å for 243 Ca atoms. The structural characterization in this study was performed using chain A unless otherwise specified. Analyses of the interaction surfaces using the protein interactions, surfaces and assemblies (PISA) server yielded the average surface area of 10,728 Å2 of monomers. The tetramer interface is more extensive. The surface area of the tetramer (ABCD) is 37,090 Å2. The PISA analysis on the interaction surfaces indicates that the tetramer

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H. Li, H. Pu / Biochemical and Biophysical Research Communications 474 (2016) 226e231

Fig. 3. Comparison of MES16 with its structural homologs. (a) Superimposition of MES16 with the above five proteins. MES16, SABP2, MKS1, BmHNL, AtHNL, and PNAE are colored in green, magenta, yellow, orange, lightpink, and palecyan, respectively. Catalytic triads of the six proteins are shown in stick representation. (B) Superimposition of the cap domains of MES16, SABP2, MKS1, BmHNL, AtHNL, and PNAE. The color scheme is same as (A). Labels correspond to the MES16 sequence.

Rauvolfia serpentine [26], R-selective hydroxynitrile lyase from A. thaliana (AtHNL) and S-selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) [27,28]. The amino-acid sequence identities of MES16 with them are above 31%. Secondary structure alignments were produced using ESPript3.0 (Fig. 2). MES16 differs from SABP2, MKS1, PNAE, AtHNL and BmHNL by 2.0, 1.6, 1.8, 1.7, and 1.6 Å (r.m.s.d. values) over the equivalent Ca atoms, respectively. While all these proteins possess a truncated a/ b-hydrolase fold and a cap domain insertion after structural superimposition, the major difference between MES16 and the other proteins lies at the cap domain. Although the two a-helices (a4 and a5) of MES16 are almost structurally conserved with other proteins, two b-sheets (b5, b6) and the loop between them are obviously divergent (Fig. 3B). Moreover, the catalytic residues of a/b-hydrolases typically lie in highly conserved core regions. The Ser-His-Asp catalytic triad is conserved in MES16, SABP2, AtHNL and BmHNL, except for MKS1 and PNAE, which has catalytic triad of Ala-His-Asn and Ser-His-Ala, respectively (Fig. 3A). 3.4. Esterase activity of MES16 Fig. 4. HPLC analysis of MES16 (black) and MES16 Ser87Ala mutant (purple) with MeIAA as substrate. The peak site of IAA is at 3.2 min and the peak site of MeIAA is at 4.7 min.

arrangement is also likely to be present in solution. This was verified by our gel filtration chromatography result (Fig. S2). In the crystal structure, residues 119e135 containing an 8e10residue a-helix (denoted aD) and part of loop are missing, probably due to their flexibility. MES16 can be divided into two domains. The core domain contains a central six-stranded parallel b-sheet (named b1-b4, b7 and b8) that is flanked on both sides by five helices (a1-a3, a7 and a8). The cap domain contains a two-stranded antiparallel b-sheet (b5 and b6) and three a helices (a4-a6) (Fig. 1C). 3.3. Structure homologs of MES16 We performed a structural similarity search with the DALI server (Table 2) [24]. The DALI results revealed that MES16 is mostly similar to these five proteins: Methyl salicylate esterase SABP2 from N. tabacum [23], Methylketone synthase1 (MKS1) from Solanum habrochaites [25], Polyneuridine aldehyde esterase (PNAE) from

Because MES16 displays MeIAA hydrolase activity in vitro [16,17], we performed an in vitro esterase analysis of MES16. MES16 display an obvious MeIAA hydrolase activity at pH 7.5. As a member of the a/b-hydrolase superfamily, the active site of MES16 is defined by the presence of a catalytic triad, Ser-87, His-239, and Asp-211. To confirm that Ser-87 of the catalytic triad is essential for MES16's esterase activity, we created the Ser87Ala mutant. Expectly, The Ser87Ala protein has no esterase activity on MeIAA (Fig. 4). 4. Discussion In this paper, we determined the crystal structure of Arabidopsis MES16 at 2.8 Å resolution. MES16 is a member of the a/b-hydrolase superfamily, with Ser-87, His-239, and Asp-211 as the catalytic triad. The Ser-87 is located in the sharp turn between strand b3 and helix a3 of the core domain, with a strained main-chain conformation. His-239 is located in the loop connecting strand b8 and h5, and Asp-211 is located in the loop connecting strand b7 and helix a7. The cap domain especially strands b5 and b6, and helices a4 and a5, covers the exposed side of the active site (Fig. 1C). MES16 and its five homolog proteins show high sequence identity, and they all possess an a/b-hydrolase fold and a cap

H. Li, H. Pu / Biochemical and Biophysical Research Communications 474 (2016) 226e231

domain insertion. These cap domains of MSK1 and BmHNL are reported to contributing the central tunnel and the specific positioning of the two helices (a6 and aD) is responsible in part for the tunnel size [25,28]. Moreover, residues at the loop region in MSK1 and hydroxynitrile lyase from Manihot esculenta have been implicated in conferring substrate specificity [25,29]. MES16 was reported demethylate both Pheide and pFCC, and also been shown to hydrolyze MeIAA and MeJA in vitro [16,17]. So, residues 119e135 containing the helix aD and part of the loop which were absent in the structure of MES16 may be related with its diverse substrates.

[10]

[11] [12]

[13]

[14]

Acknowledgments The authors greatly thank the staff at Shanghai Synchrotron Radiation Facility (SSRF) for diffraction data collection. This work was supported by the National Natural Science Foundation of China (Grant No. 31300634).

[15]

[16]

[17]

Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.115. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.04.115.

[18] [19]

[20]

[21]

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