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Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l -norleucine
BBA - Proteins and Proteomics 1865 (2017) 1123–1128
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Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with L-norleucine☆
MARK
Svetlana V. Revtovich, Elena A. Morozova, Vitalia V. Kulikova, Natalya V. Anufrieva, Tatyana I. Osipova, Vasiliy S. Koval, Alexey D. Nikulin1, Tatyana V. Demidkina⁎,1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords: Pyridoxal 5′-phosphate-dependent methionine γ-lyase C115H mutant form Three-dimensional structure
The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-L-homoserine and in the β-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, L-norleucine was determined at 1.45 Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and β-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.
1. Introduction Pyridoxal 5′-phosphate (PLP)-dependent methionine γ-lyase (MGL, EC 4.4.1.11) catalyzes the γ- and β-elimination reactions of methionine and S-substituted analogues of cysteine to produce α-keto acids, alkylthiols and ammonia (Scheme 1) [1,2]. Besides, MGL catalyzes the γ-replacement reactions of L-methionine derivatives and the β-replacement reactions of L-cysteine and S-substituted L-cysteines [1,2]. Biochemical properties of MGL from bacteria, primitive eukaryotic pathogens, and plants, its putative roles in living cell, and an exploiting of the enzyme for the treatment of cancers and as a target against microbial infections have been reviewed in [5–7]. Previously, we showed that recombinant MGL from Citrobacter freundii and the mutant form with the replacement of Cys115 for Ala (C115A MGL) catalyze the β-elimination reaction of (2R)-2-amino-3[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) to form well known antibacterial thiosulfinate from garlic, 2-propenethiosulfinate (allicin) [8]. Then, the production of other thiosulfinates in the γ- and β-elimination reactions of sulfoxides — analogues of cysteine and methionine by MGL (Supplementary Scheme S1) and antibacterial activity of the mixtures of the C. freundii and Clostridium sporogenes MGLs with sulfoxides was demonstrated [9–11]. This opens a way to the rational
design of a new antimicrobial drug producer. A drawback of this way is that the reactions were followed by the inactivation of the wild type and the mutant enzyme in the γ- and β-elimination reactions. X-ray data on the inactivated wild type and C115A MGL demonstrated that the interaction of MGL with alliin led to the oxidation of three SH-groups of the enzyme, surface Cys4, active site Cys115, and buried Cys245 in the wild type enzyme and two SH-groups, Cys4 and Cys245 in the C115A MGL. As a result, the disturbance of H-bonds essential for the catalysis of the γ-elimination reaction in the triad Cys115/Tyr113/Arg60 leads to the wild type and C115A MGL becoming inactivated in the γ-elimination reaction by 74% and 63%. Meanwhile, the inactivation of the enzyme in the catalysis of the βelimination reaction was shown to be 31% for the wild type enzyme and 13% for the C115A MGL. We supposed that in the case of the wild type MGL the retardation of the β-elimination reaction is mainly due to the steric hindrances for a substrate accommodation induced by the lengthening of Cys115 side chain with the allyl radical [8]. To have an efficient catalyst in the β-elimination reaction of sulfoxides (analogues of S-alk(en)yl-substituted cysteines), a mutant form of the enzyme with a replacement of Cys115 proved to be the preferred embodiment. The active site cysteine residue is highly conservative in MGL from different sources [12]. A study of 19 mutant forms of Pseudomonas putida MGL with the replacements of cysteine 116 showed its
Abbreviations: MGL, methionine γ-lyase; PEG MME, polyethylene glycol monomethyl ether; PLP, pyridoxal 5′-phosphate ☆ Dedicated to the 115th anniversary of academician A. E. Braunstein's birth. ⁎ Corresponding author. E-mail address: [email protected] (T.V. Demidkina). 1 Equal contribution. http://dx.doi.org/10.1016/j.bbapap.2017.06.001 Received 16 January 2017; Received in revised form 2 June 2017; Accepted 5 June 2017 Available online 06 June 2017 1570-9639/ © 2017 Elsevier B.V. All rights reserved.
BBA - Proteins and Proteomics 1865 (2017) 1123–1128
S.V. Revtovich et al.
Lys210 R
S
n= 1; 2
R
COO-
(CH2)n
NH3+
+
COO-
(CH 2)n
R
NH+
NH PLP
PLP internal aldimine λmax ~ 420 nm
COO-
(CH 2)n
S
+
NH
+
S
COOβ-elimination RSH α-aminoacrylate λmax ~ 460-480 nm
quinonoid intermediate λmax ~ 500 nm
external aldimine λmax ~ 420 nm
NH
MGL +
+
PLP
PLP
NH4+ CH3
COO-
O pyruvate λmax ~ 320 nm
γ-elimination
NH4+
MGL
CH3
R
PLP
R
COO-
S
NH+
NH PLP
RSH
α-aminocrotonate λmax ~ 460-480 nm
COO-
S
+
NH
COO-
O α-ketobutyrate λmax ~ 320 nm
COO-
CH 3
+
PLP
enamine λmax ~ 320 nm
ketimine intermediate λmax ~ 320 nm
Scheme 1. Chemical mechanisms of PLP-dependent β- and γ-elimination reactions [3,4].
importance for the γ-elimination reaction of methionine to proceed. At the same time, it was found that the mutant form of P. putida MGL with the replacement of Cys116 for His possessed the catalytic efficiency more than ten times higher than wild type enzyme in the β-elimination reaction [13,14]. We have prepared and studied mutant forms of С. freundii and C. sporogenes MGL with the replacement of active site cysteine 115 with histidine (C115H MGL). The mutant enzymes decomposed S-alk(en)ylsubstituted cysteines more effectively than the wild type enzymes [10,11]. However, they proved to be inactive in the γ-elimination reaction of physiological substrate. Here, we present the crystal structure of C. freundii C115H MGL complexed with a competitive inhibitor, Lnorleucine, and analyze structural peculiarities leading to the deleterious effect of the C115H replacement on the γ-elimination reaction.
Table 1 Data collection and refinement statistics. Values in parentheses are for the highest resolution shell. Space group
I222
Unit cell parameters (Å)
a = 56.51, b = 123.02, с = 127.58 α = β = γ = 90° 0.95372 88.56–1.45 (1.47–1.45) 99.1 (95.1) 4.40 (4.20) 0.054 (0.800) 0.061 (1.136) 14.9 (1.3) 0.999 (0.569) 1, 2, 398 2992 346 74,318 0.128/0.169 (0.289/0.318) 3805 (5.12%) 26.21
Wavelength (Å) Resolution (Å) Completeness (%) Redundancy Rmerge (%) Rmeas (%) Mean((I)/sd(I)) CC(1/2) Disordered protein residues No. of non-H protein atoms No. of water atoms No. of unique reflections R/Rfree Rfree test set reflections Mean temperature factor B (Å2) R.m.s. deviation from ideal values Bond lengths (Å) Bond angles (°) Chirality angles (°) Planar angles (°) Ramachandran plot Favoured region (%) Allowed region (%) Outlier region (%)
2. Materials and methods 2.1. Crystallization and data collection The mutant form, C115H MGL, was isolated and purified as described earlier [11]. Crystals of C115H MGL were obtained at the same conditions as described in [15] using 37.5% polyethylene glycol monomethyl ether (PEG MME) 2000 as the precipitant. Separate rhombic crystals appeared after a week and grew to 0.3 mm within ten days. L-Norleucine was demonstrated to be a competitive inhibitor of the enzyme with Ki = 0.6 mM [16]. The complex of C115H MGL with Lnorleucine was obtained by soaking of holoenzyme crystals in the cryoprotective mother liquid solution containing L-norleucine (12 mM) during 20 min. Diffraction data were collected at the European Synchrotron (ESRF) beamline ID29 (Grenoble, France) using Pilatus 6M (Dectris) detector and processed by the XDS program [17]. The crystals belonged to space group I222 with unit-cell parameters a = 56.51, b = 123.02, and c = 127.58 Å and contained one monomer in the asymmetric unit. The detailed data collecting statistics are shown in Table 1.
0.020 1.833 0.130 0.010 98.05 1.71 0.24 [Ser190]†
CC1/2 is the Pearson's correlation coefficient calculated for Imean by splitting the data randomly in half by AIMLESS/SCALA [20]. † The conformation of the amino-acid residue at this position is a characteristic feature of the protein [15].
bias during the first round of refinement. According to the mFo-DFc electron density simulated-annealing omit map, we localized L-norleucine at the active site of the enzyme covalently bound with the cofactor (Supplementary Fig. S1A). Subsequent refinement and rebuilding with σA-weighted 2mFo-DFc and mFo-DFc density maps allowed us to recognize the alternative position of L-norleucine inside the active site unbound to the cofactor (Supplementary Fig. S1B). An excess of electron density at the sulfur atom of Cys4 forced us to change this residue to 3-sulfenoalanine. The final model, refined to 1.45 Å, includes 3508 non-hydrogen atoms. It includes the external aldimine (Supplementary Fig. S1C), Lnorleucine, PEG MME 2000 molecule and 346 water molecules converged to Rwork of 12.8% and Rfree of 16.9% for the data between
2.2. Structure determination and refinement The structure was solved by molecular replacement using the structure of C. freundii MGL (PDB ID: 2RFV) by rigid body procedure, implemented in the CCP4 software suite [18]. The model was improved using manual rebuilding with COOT [19] and maximum likelihood refinement using REFMAC5 [18]. Flexible loops of the protein and water molecules were removed from the initial model to exclude model 1124
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is composed of two subunits related by a 2-fold axis. The replacement did not influence the overall polypeptide chain fold of a subunit (Fig. 1) and intersubunit contacts in a catalytic dimer as compared to those of the wild type enzyme. Crystal structures of wild type C. freundii MGL holoenzyme contain two flexible regions, N-terminal (residues 47–63) and C-terminal (residues 353–368). The N-terminal mobile region belongs to the long part of the N-terminal domain and includes residues 47–63. Side chains of Phe49, Ile57 and Leu61 compose a part of a methionine-binding hydrophobic pocket and side groups of Arg60 and Tyr58 are involved in the binding of the phosphate “handle” of the cofactor. Binding of substrates or inhibitors in the active site leads to a stabilization of the Nterminal region [21,22]. A restriction of flexibility of this part is observed in the structure of the C115H MGL complexed with L-norleucine (Fig. 1), that confirms our suggestion on the significance of the Nterminal part stabilization for the catalysis of the γ- and β-elimination reactions [21]. Another mobile region (residues 353–368) belongs to the C-terminal domain and is located at the entrance into an active site cavity. B-factors of this region are about 64 Å2 (the structure of С115H MGL, 1.45 Å resolution), 49 Å2 (the holoenzyme structure PDB ID: 2RFV, 1.35 Å resolution), and 80 Å2 (the holoenzyme structure PDB ID: 1Y4I, 1.90 Å resolution). Such variability of average B-factors of residues 353–368 was observed in all known C. freundii MGL structures as well. We suggested that the mobility of this region may be necessary to provide a way out of the products of the γ- and β-elimination reactions [22]. 3.2. The enzyme active site Fig. 1. Superposition of the Cα traces in crystal structures of C. freundii wild type MGL (PDB ID: 2RFV) and complex of C115H MGL with L-norleucine (PDB ID: 5M3Z) colorized by B-factor value increased from dark blue to red.
We observed two alternative positions of L-norleucine inside of the C115H MGL active site (Fig. 2). In one of them, L-norleucine is close to the cofactor but does not form the aldimine bond with it (Fig. 3A). The distance from the nitrogen atom of the amino group of L-norleucine to the C4′ atom of PLP is 3.08 Å. PLP is covalently linked to the ε-amino group of Lys210. This structure might be considered as modeling the Michaelis complex. The position of L-norleucine is stabilized by electrostatic interactions and Hbonds, which it forms with residues of the active site. An oxygen atom of the L-norleucine carboxylate group is within H-bond distance to the NH2 atom of the Arg374 guanidinium group (3.09 Å) and the nitrogen atom of the Ser339 main chain (3.10 Å). The nitrogen atom of the Lnorleucine amino group is within H-bond distance of the oxygen atoms of Tyr113 (2.61 Å) and Tyr58* (3.35 Å) side chains. Two water molecules W501 and W530 form the H-bond network between Asn160,
88.56 Å and 1.45 Å (Table 1). The structure has been submitted to the Protein Data Bank with PDB ID: 5M3Z. 3. Results and discussion 3.1. The overall structure The three-dimensional structure of the C115H MGL complex with Lnorleucine, the competitive inhibitor of the enzyme, a structural analog of methionine, was solved and refined at 1.45 Å resolution. Like the wild type enzyme the complex exists as a tetramer which is composed of two catalytic dimers linked with a 2-fold symmetry axis. Each dimer
Fig. 2. Stereo view of the C115H MGL active site in complex with L-norleucine. Residues of the neighboring subunit of a catalytic dimer are marked with asterisks. The alternative conformation is shown in cyan.
1125
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Fig. 3. The C115H MGL active site. H-bond networks in (A) the Michaelis complex and (B) the external aldimine are shown. Residues of a neighboring subunit of a catalytic dimer are marked with asterisks.
R374, L-norleucine and the Ο3′-atom of PLP. In the second position, L-norleucine is covalently bound to the cofactor, thus forming the external aldimine (Fig. 3B; Scheme 1). Its binding is provided by the carboxylate group, which is involved in Hbond interactions with NH1 and NH2 atoms of the Arg374 guanidinium group and in H-bond with the main chain nitrogen atom of Ser339. The side chain of L-norleucine is stabilized by hydrophobic interactions with Phe49*, Ile57* and Leu61*. The main difference between the active site structures of the Michaelis complex and the external aldimine is simultaneous tilts of the PLP and Tyr113 rings observed in the structure of the external aldimine and free side chain of Lys210. As a result of reciprocal tilts of PLP and Tyr113 rings ongoing from the structure of Michaelis complex to the structure of the external aldimine, the side chain of Tyr113 draws near the ND atom of His115 (Fig. 3). This movement is analogous to that observed coming out of the structure of the wild type holoenzyme to the structure of the external aldimine with glycine [21]. The position of His115 is restricted by Tyr113 (bottom), Lys239* (top) and Arg60* (laterally). The position of the residue is close to its position in two crystal structures of the Michaelis complexes of the mutant form P. putida MGL with the replacement of homologous cysteine 116 for histidine [14]. Nevertheless, in three structures the position of the imidazole ring is different due to a rotation of the rings around the χ2 angle. For the enzymes of the cystathionine β-lyase subclass, the active site lysine residue was proposed to be a general acid catalyst in the β- and γelimination reactions [4]. Analysis of crystal structures of the external aldimine of C. freundii MGL with glycine [21] and the complex of the enzyme with L-cycloserine modeling ketimine intermediate allowed us to suggest that Lys210 performs the 1,3-prototropic shift of the Cαproton to the C4′-atom of the cofactor and serves as a base abstracting β-protons from a number of inhibitors and physiological substrate [22]. In the structure under consideration, the mutual location of L-norleucine and active site residues is like that in the external aldimine with glycine. The position of the Lys210 amino group at 3.03 Å to the Cαatom of L-norleucine is provided by interactions with side chains of Ser339 (2.85 Å) and Tyr 58 (2.61 Å). It is at 3.40 Å from the Cβ-atom of L-norleucine. The structure confirms the suggestion that the interaction of Lys210 with Tyr58 is necessary to provide optimal positioning of Lys210 at the stage of Cα-proton abstraction from an external aldimine and at some other stages of the β- and γ-elimination reactions [23]. Like in the other structures of the MGL complexes with amino acids,
the carbonyl group of the peptide bond between Thr338 and Ser339 residues turns by 180° relative to its position in wild type holoenzyme. It is necessary for the accommodation of amino acids at the active site [21,24]. 3.3. The γ- and β-elimination reactions catalyzed by C115H MGL Kinetic and spectral studies of C115H MGL demonstrated that the mutant enzyme did not catalyze the γ-elimination reaction of the physiological substrate but catalyzed the γ-elimination reaction of Oacetyl-L-homoserine with a kcat/Km value increased by one order of magnitude compared to that for the wild type enzyme. Catalytic efficiency of C115H MGL in the β-elimination reaction of S-alkylcysteines proved to be comparable to that for the wild type MGL [11]. Thus the replacement of Cys115 for histidine did not change catalytic properties of the enzyme in the β-elimination reaction. Obviously in the case of the γ-elimination reaction, the replacement did not disturb neither the stage of transaldimination nor the stage of Cα-proton abstraction (Scheme 1) which are common for the β- and γelimination reactions. In the spectrum of the C115H mutant enzyme complexed with methionine, the band belonging to the aminocrotonate is absent [11] while it is present in the analogous spectrum of the wild type MGL [16]. Hence the replacement interrupts the stage of methythiol elimination. The mutant form of P. putida MGL with the replacement of homologous cysteine 116 by histidine catalyzed the γ-elimination reaction of methionine with catalytic efficiency decreased for about four orders of magnitude compared to that of wild type enzyme. Meanwhile, our results [11] are not corresponding to the increased catalytic efficiency of the mutant form of P. putida MGL with the replacement of cysteine 116 in the β-elimination reaction [13,14]. The elimination of a γ-substituent needs an acid catalyst to proceed. A dual role was proposed for the active site tyrosine residue (Tyr113 of C. freundii MGL) conserved in the family of PLP-dependent enzymes of the cystathionine β-lyase subclass [4,25,26]. It was proposed that it acts as a base, abstracting a proton from an amino group of the incoming substrate to allow its nucleophilic attack on the C4′-atom of the internal aldimine [4,25]. Data on the pH-dependence of the γ-elimination reaction of methionine catalyzed by C. freundii MGL [27] and the spatial structures of MGL complexes with amino acids modeling Michaelis complexes [24] suggests that Tyr113 fulfills this role. This residue was proposed to be a general acid catalyst in the γ-elimination reaction 1126
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catalyzed by the enzymes of the cystathionine β-lyase subclass [4,25,26]. The replacement of Tyr114 of P. putida MGL by Phe gave evidence that the residue might perform acidic catalysis at the stage of methylthiol elimination [28]. The analogous mutant form of C. freundii with the replacement of Tyr113 by Phe catalyzed the γ-elimination of methionine with the catalytic efficiency reduced by four orders of magnitude as well (unpublished data). Comparison of the spatial structures of the C. freundii [15] and P. putida [29] MGLs, the structures of P. putida MGL C116H mutant form complexed with two substrates [14] and the structure of the external aldimine of C. freundii MGL with glycine [21] revealed the network of H-bonding contacts between conservative residues Tyr113, Cys115, Lys239, Asp240 (the numeration for C. freundii MGL) in MGL from different bacteria. The contacts become shorter on going from the structure of C. freundii MGL holoenzyme to the structure of the external aldimine with glycine [21]. This led us to propose that the proton abstracted from an amino group of a substrate could be stored at Cys115 or at Asp240 and then could be returned to Tyr113 to catalyze the stage of methylthiol elimination [8]. It was discussed above that the catalytic properties of the C115H mutant enzyme are evidence for which the mutant form catalyzes the stage of transaldimination, i.e. in the reactions of C115H enzyme with substrates of the γ- and β-elimination reactions, the Tyr113 hydroxylic group acts as a base. In the structure of the Michaelis complex, the nitrogen atom of the amino group of L-norleucine is 3.08 Å from the C4′ atom of the cofactor, thus making it possible to perform a nucleoрhilic attack on it. Spectral data on the C115H holoenzyme revealed that the main ionic form of the internal aldimine is a ketoenamine, i.e. the form with a positively charged aldimine nitrogen [11]. A nucleophility of the L-norleucine amino group may be provided by its closeness to the positive charge of the nitrogen atom of the ketoenamine. It is possible that the proton abstracted from the amino group of L-norleucine remains at the Tyr113 hydroxylic group as a short H-bond to the amino group of Lnorleucine holds it back. The closeness of the hydroxylic group of Tyr113 to the imidazole group of His115 creates favorable conditions for the formation of an Hbond between these groups. The pKa value of Tyr113 in the wild type enzyme was estimated at 7.1–7.2 [27], which is pretty close to the pKa of imidazole. Thus, even the possibility of formation of a low-barrier Hbond may be considered under certain circumstances. If such interactions take place at the stage of methylthiol elimination, Tyr113 should be unable to be a general acid catalyst because its hydroxylic proton is involved in the direct interaction with His115. These considerations may explain the absence of the γ-elimination reaction for C115H MGL. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2017.06.001.
[4]
[5]
[6]
[7]
[8]
[9]
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[17] [18]
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[21]
Transparency Document [22]
The Transparency document associated with this article can be found, in online version. Acknowledgements
[23]
This work was supported by the Russian Science Foundation (project No. 15-14-00009). We thank Dr. Nicolai G. Faleev for critical reading of the manuscript.
[24]
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Demidkina, Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and β-protons of competitive inhibitors, Biochimie 101 (2014) 161–167, http://dx.doi.org/10.1016/j.biochi.2014.01.007. N.A. Kuznetsov, N.G. Faleev, A.A. Kuznetsova, E.A. Morozova, S.V. Revtovich, N.V. Anufrieva, A.D. Nikulin, O.S. Fedorova, T.V. Demidkina, Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors, J. Biol. Chem. 290 (2015) 671–681, http://dx.doi.org/10.1074/jbc.M114. 586511. N.V. Anufrieva, N.G. Faleev, E.A. Morozova, N.P. Bazhulina, S.V. Revtovich, V.P. Timofeev, Y.V. Tkachev, A.D. Nikulin, T.V. Demidkina, The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase, Biochim. Biophys. Acta 1854 (2015) 1220–1228, http://dx.doi.org/10.1016/j.bbapap.2014.12.027. S.V. Revtovich, E.A. Morozova, E.N. Khurs, L.N. Zakomirdina, A.D. Nikulin, T.V. Demidkina, R.M. Khomutov, Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates, Biochem. Mosc. 76 (2011) 564–570, http://dx.doi.org/10.1134/S0006297911050063. T. Clausen, R. Huber, B. Laber, H.D. Pohlenz, A. Messerschmidt, Crystal structure of the pyridoxal-5′-phosphate dependent cystathionine β-lyase from Escherichia coli at 1.83 Å, J. Mol. Biol. 262 (1996) 202–224, http://dx.doi.org/10.1006/jmbi.1996.0508. T. Clausen, R. Huber, L. Prade, M.C. Wahl, A. Messerschmidt, Crystal structure of Escherichia coli cystathionine γ-synthase at 1.5 Å resolution, EMBO J. 23 (1998) 6827–6838, http://dx.doi.org/10.1093/emboj/17.23.6827. N. Faleev, K. Alferov, M. Tsvetikova, E. Morozova, S. Revtovich, E. Khurs, M. Vorob'ev, R. Phillips, T. Demidkina, R. Khomutov, Methionine γ-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity,
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[29] H. Motoshima, K. Inagaki, T. Kumasaka, M. Furuichi, H. Inoue, T. Tamura, N. Esaki, K. Soda, N. Tanaka, M. Yamamoto, H. Tanaka, Crystal structure of the pyridoxal 5′phosphate dependent L-methionine gamma-lyase from Pseudomonas putida, J. Biochem. 128 (2000) 349–354.
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Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with L-norleucine☆
MARK
Svetlana V. Revtovich, Elena A. Morozova, Vitalia V. Kulikova, Natalya V. Anufrieva, Tatyana I. Osipova, Vasiliy S. Koval, Alexey D. Nikulin1, Tatyana V. Demidkina⁎,1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords: Pyridoxal 5′-phosphate-dependent methionine γ-lyase C115H mutant form Three-dimensional structure
The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-L-homoserine and in the β-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, L-norleucine was determined at 1.45 Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and β-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.
1. Introduction Pyridoxal 5′-phosphate (PLP)-dependent methionine γ-lyase (MGL, EC 4.4.1.11) catalyzes the γ- and β-elimination reactions of methionine and S-substituted analogues of cysteine to produce α-keto acids, alkylthiols and ammonia (Scheme 1) [1,2]. Besides, MGL catalyzes the γ-replacement reactions of L-methionine derivatives and the β-replacement reactions of L-cysteine and S-substituted L-cysteines [1,2]. Biochemical properties of MGL from bacteria, primitive eukaryotic pathogens, and plants, its putative roles in living cell, and an exploiting of the enzyme for the treatment of cancers and as a target against microbial infections have been reviewed in [5–7]. Previously, we showed that recombinant MGL from Citrobacter freundii and the mutant form with the replacement of Cys115 for Ala (C115A MGL) catalyze the β-elimination reaction of (2R)-2-amino-3[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) to form well known antibacterial thiosulfinate from garlic, 2-propenethiosulfinate (allicin) [8]. Then, the production of other thiosulfinates in the γ- and β-elimination reactions of sulfoxides — analogues of cysteine and methionine by MGL (Supplementary Scheme S1) and antibacterial activity of the mixtures of the C. freundii and Clostridium sporogenes MGLs with sulfoxides was demonstrated [9–11]. This opens a way to the rational
design of a new antimicrobial drug producer. A drawback of this way is that the reactions were followed by the inactivation of the wild type and the mutant enzyme in the γ- and β-elimination reactions. X-ray data on the inactivated wild type and C115A MGL demonstrated that the interaction of MGL with alliin led to the oxidation of three SH-groups of the enzyme, surface Cys4, active site Cys115, and buried Cys245 in the wild type enzyme and two SH-groups, Cys4 and Cys245 in the C115A MGL. As a result, the disturbance of H-bonds essential for the catalysis of the γ-elimination reaction in the triad Cys115/Tyr113/Arg60 leads to the wild type and C115A MGL becoming inactivated in the γ-elimination reaction by 74% and 63%. Meanwhile, the inactivation of the enzyme in the catalysis of the βelimination reaction was shown to be 31% for the wild type enzyme and 13% for the C115A MGL. We supposed that in the case of the wild type MGL the retardation of the β-elimination reaction is mainly due to the steric hindrances for a substrate accommodation induced by the lengthening of Cys115 side chain with the allyl radical [8]. To have an efficient catalyst in the β-elimination reaction of sulfoxides (analogues of S-alk(en)yl-substituted cysteines), a mutant form of the enzyme with a replacement of Cys115 proved to be the preferred embodiment. The active site cysteine residue is highly conservative in MGL from different sources [12]. A study of 19 mutant forms of Pseudomonas putida MGL with the replacements of cysteine 116 showed its
Abbreviations: MGL, methionine γ-lyase; PEG MME, polyethylene glycol monomethyl ether; PLP, pyridoxal 5′-phosphate ☆ Dedicated to the 115th anniversary of academician A. E. Braunstein's birth. ⁎ Corresponding author. E-mail address: [email protected] (T.V. Demidkina). 1 Equal contribution. http://dx.doi.org/10.1016/j.bbapap.2017.06.001 Received 16 January 2017; Received in revised form 2 June 2017; Accepted 5 June 2017 Available online 06 June 2017 1570-9639/ © 2017 Elsevier B.V. All rights reserved.
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Lys210 R
S
n= 1; 2
R
COO-
(CH2)n
NH3+
+
COO-
(CH 2)n
R
NH+
NH PLP
PLP internal aldimine λmax ~ 420 nm
COO-
(CH 2)n
S
+
NH
+
S
COOβ-elimination RSH α-aminoacrylate λmax ~ 460-480 nm
quinonoid intermediate λmax ~ 500 nm
external aldimine λmax ~ 420 nm
NH
MGL +
+
PLP
PLP
NH4+ CH3
COO-
O pyruvate λmax ~ 320 nm
γ-elimination
NH4+
MGL
CH3
R
PLP
R
COO-
S
NH+
NH PLP
RSH
α-aminocrotonate λmax ~ 460-480 nm
COO-
S
+
NH
COO-
O α-ketobutyrate λmax ~ 320 nm
COO-
CH 3
+
PLP
enamine λmax ~ 320 nm
ketimine intermediate λmax ~ 320 nm
Scheme 1. Chemical mechanisms of PLP-dependent β- and γ-elimination reactions [3,4].
importance for the γ-elimination reaction of methionine to proceed. At the same time, it was found that the mutant form of P. putida MGL with the replacement of Cys116 for His possessed the catalytic efficiency more than ten times higher than wild type enzyme in the β-elimination reaction [13,14]. We have prepared and studied mutant forms of С. freundii and C. sporogenes MGL with the replacement of active site cysteine 115 with histidine (C115H MGL). The mutant enzymes decomposed S-alk(en)ylsubstituted cysteines more effectively than the wild type enzymes [10,11]. However, they proved to be inactive in the γ-elimination reaction of physiological substrate. Here, we present the crystal structure of C. freundii C115H MGL complexed with a competitive inhibitor, Lnorleucine, and analyze structural peculiarities leading to the deleterious effect of the C115H replacement on the γ-elimination reaction.
Table 1 Data collection and refinement statistics. Values in parentheses are for the highest resolution shell. Space group
I222
Unit cell parameters (Å)
a = 56.51, b = 123.02, с = 127.58 α = β = γ = 90° 0.95372 88.56–1.45 (1.47–1.45) 99.1 (95.1) 4.40 (4.20) 0.054 (0.800) 0.061 (1.136) 14.9 (1.3) 0.999 (0.569) 1, 2, 398 2992 346 74,318 0.128/0.169 (0.289/0.318) 3805 (5.12%) 26.21
Wavelength (Å) Resolution (Å) Completeness (%) Redundancy Rmerge (%) Rmeas (%) Mean((I)/sd(I)) CC(1/2) Disordered protein residues No. of non-H protein atoms No. of water atoms No. of unique reflections R/Rfree Rfree test set reflections Mean temperature factor B (Å2) R.m.s. deviation from ideal values Bond lengths (Å) Bond angles (°) Chirality angles (°) Planar angles (°) Ramachandran plot Favoured region (%) Allowed region (%) Outlier region (%)
2. Materials and methods 2.1. Crystallization and data collection The mutant form, C115H MGL, was isolated and purified as described earlier [11]. Crystals of C115H MGL were obtained at the same conditions as described in [15] using 37.5% polyethylene glycol monomethyl ether (PEG MME) 2000 as the precipitant. Separate rhombic crystals appeared after a week and grew to 0.3 mm within ten days. L-Norleucine was demonstrated to be a competitive inhibitor of the enzyme with Ki = 0.6 mM [16]. The complex of C115H MGL with Lnorleucine was obtained by soaking of holoenzyme crystals in the cryoprotective mother liquid solution containing L-norleucine (12 mM) during 20 min. Diffraction data were collected at the European Synchrotron (ESRF) beamline ID29 (Grenoble, France) using Pilatus 6M (Dectris) detector and processed by the XDS program [17]. The crystals belonged to space group I222 with unit-cell parameters a = 56.51, b = 123.02, and c = 127.58 Å and contained one monomer in the asymmetric unit. The detailed data collecting statistics are shown in Table 1.
0.020 1.833 0.130 0.010 98.05 1.71 0.24 [Ser190]†
CC1/2 is the Pearson's correlation coefficient calculated for Imean by splitting the data randomly in half by AIMLESS/SCALA [20]. † The conformation of the amino-acid residue at this position is a characteristic feature of the protein [15].
bias during the first round of refinement. According to the mFo-DFc electron density simulated-annealing omit map, we localized L-norleucine at the active site of the enzyme covalently bound with the cofactor (Supplementary Fig. S1A). Subsequent refinement and rebuilding with σA-weighted 2mFo-DFc and mFo-DFc density maps allowed us to recognize the alternative position of L-norleucine inside the active site unbound to the cofactor (Supplementary Fig. S1B). An excess of electron density at the sulfur atom of Cys4 forced us to change this residue to 3-sulfenoalanine. The final model, refined to 1.45 Å, includes 3508 non-hydrogen atoms. It includes the external aldimine (Supplementary Fig. S1C), Lnorleucine, PEG MME 2000 molecule and 346 water molecules converged to Rwork of 12.8% and Rfree of 16.9% for the data between
2.2. Structure determination and refinement The structure was solved by molecular replacement using the structure of C. freundii MGL (PDB ID: 2RFV) by rigid body procedure, implemented in the CCP4 software suite [18]. The model was improved using manual rebuilding with COOT [19] and maximum likelihood refinement using REFMAC5 [18]. Flexible loops of the protein and water molecules were removed from the initial model to exclude model 1124
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is composed of two subunits related by a 2-fold axis. The replacement did not influence the overall polypeptide chain fold of a subunit (Fig. 1) and intersubunit contacts in a catalytic dimer as compared to those of the wild type enzyme. Crystal structures of wild type C. freundii MGL holoenzyme contain two flexible regions, N-terminal (residues 47–63) and C-terminal (residues 353–368). The N-terminal mobile region belongs to the long part of the N-terminal domain and includes residues 47–63. Side chains of Phe49, Ile57 and Leu61 compose a part of a methionine-binding hydrophobic pocket and side groups of Arg60 and Tyr58 are involved in the binding of the phosphate “handle” of the cofactor. Binding of substrates or inhibitors in the active site leads to a stabilization of the Nterminal region [21,22]. A restriction of flexibility of this part is observed in the structure of the C115H MGL complexed with L-norleucine (Fig. 1), that confirms our suggestion on the significance of the Nterminal part stabilization for the catalysis of the γ- and β-elimination reactions [21]. Another mobile region (residues 353–368) belongs to the C-terminal domain and is located at the entrance into an active site cavity. B-factors of this region are about 64 Å2 (the structure of С115H MGL, 1.45 Å resolution), 49 Å2 (the holoenzyme structure PDB ID: 2RFV, 1.35 Å resolution), and 80 Å2 (the holoenzyme structure PDB ID: 1Y4I, 1.90 Å resolution). Such variability of average B-factors of residues 353–368 was observed in all known C. freundii MGL structures as well. We suggested that the mobility of this region may be necessary to provide a way out of the products of the γ- and β-elimination reactions [22]. 3.2. The enzyme active site Fig. 1. Superposition of the Cα traces in crystal structures of C. freundii wild type MGL (PDB ID: 2RFV) and complex of C115H MGL with L-norleucine (PDB ID: 5M3Z) colorized by B-factor value increased from dark blue to red.
We observed two alternative positions of L-norleucine inside of the C115H MGL active site (Fig. 2). In one of them, L-norleucine is close to the cofactor but does not form the aldimine bond with it (Fig. 3A). The distance from the nitrogen atom of the amino group of L-norleucine to the C4′ atom of PLP is 3.08 Å. PLP is covalently linked to the ε-amino group of Lys210. This structure might be considered as modeling the Michaelis complex. The position of L-norleucine is stabilized by electrostatic interactions and Hbonds, which it forms with residues of the active site. An oxygen atom of the L-norleucine carboxylate group is within H-bond distance to the NH2 atom of the Arg374 guanidinium group (3.09 Å) and the nitrogen atom of the Ser339 main chain (3.10 Å). The nitrogen atom of the Lnorleucine amino group is within H-bond distance of the oxygen atoms of Tyr113 (2.61 Å) and Tyr58* (3.35 Å) side chains. Two water molecules W501 and W530 form the H-bond network between Asn160,
88.56 Å and 1.45 Å (Table 1). The structure has been submitted to the Protein Data Bank with PDB ID: 5M3Z. 3. Results and discussion 3.1. The overall structure The three-dimensional structure of the C115H MGL complex with Lnorleucine, the competitive inhibitor of the enzyme, a structural analog of methionine, was solved and refined at 1.45 Å resolution. Like the wild type enzyme the complex exists as a tetramer which is composed of two catalytic dimers linked with a 2-fold symmetry axis. Each dimer
Fig. 2. Stereo view of the C115H MGL active site in complex with L-norleucine. Residues of the neighboring subunit of a catalytic dimer are marked with asterisks. The alternative conformation is shown in cyan.
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Fig. 3. The C115H MGL active site. H-bond networks in (A) the Michaelis complex and (B) the external aldimine are shown. Residues of a neighboring subunit of a catalytic dimer are marked with asterisks.
R374, L-norleucine and the Ο3′-atom of PLP. In the second position, L-norleucine is covalently bound to the cofactor, thus forming the external aldimine (Fig. 3B; Scheme 1). Its binding is provided by the carboxylate group, which is involved in Hbond interactions with NH1 and NH2 atoms of the Arg374 guanidinium group and in H-bond with the main chain nitrogen atom of Ser339. The side chain of L-norleucine is stabilized by hydrophobic interactions with Phe49*, Ile57* and Leu61*. The main difference between the active site structures of the Michaelis complex and the external aldimine is simultaneous tilts of the PLP and Tyr113 rings observed in the structure of the external aldimine and free side chain of Lys210. As a result of reciprocal tilts of PLP and Tyr113 rings ongoing from the structure of Michaelis complex to the structure of the external aldimine, the side chain of Tyr113 draws near the ND atom of His115 (Fig. 3). This movement is analogous to that observed coming out of the structure of the wild type holoenzyme to the structure of the external aldimine with glycine [21]. The position of His115 is restricted by Tyr113 (bottom), Lys239* (top) and Arg60* (laterally). The position of the residue is close to its position in two crystal structures of the Michaelis complexes of the mutant form P. putida MGL with the replacement of homologous cysteine 116 for histidine [14]. Nevertheless, in three structures the position of the imidazole ring is different due to a rotation of the rings around the χ2 angle. For the enzymes of the cystathionine β-lyase subclass, the active site lysine residue was proposed to be a general acid catalyst in the β- and γelimination reactions [4]. Analysis of crystal structures of the external aldimine of C. freundii MGL with glycine [21] and the complex of the enzyme with L-cycloserine modeling ketimine intermediate allowed us to suggest that Lys210 performs the 1,3-prototropic shift of the Cαproton to the C4′-atom of the cofactor and serves as a base abstracting β-protons from a number of inhibitors and physiological substrate [22]. In the structure under consideration, the mutual location of L-norleucine and active site residues is like that in the external aldimine with glycine. The position of the Lys210 amino group at 3.03 Å to the Cαatom of L-norleucine is provided by interactions with side chains of Ser339 (2.85 Å) and Tyr 58 (2.61 Å). It is at 3.40 Å from the Cβ-atom of L-norleucine. The structure confirms the suggestion that the interaction of Lys210 with Tyr58 is necessary to provide optimal positioning of Lys210 at the stage of Cα-proton abstraction from an external aldimine and at some other stages of the β- and γ-elimination reactions [23]. Like in the other structures of the MGL complexes with amino acids,
the carbonyl group of the peptide bond between Thr338 and Ser339 residues turns by 180° relative to its position in wild type holoenzyme. It is necessary for the accommodation of amino acids at the active site [21,24]. 3.3. The γ- and β-elimination reactions catalyzed by C115H MGL Kinetic and spectral studies of C115H MGL demonstrated that the mutant enzyme did not catalyze the γ-elimination reaction of the physiological substrate but catalyzed the γ-elimination reaction of Oacetyl-L-homoserine with a kcat/Km value increased by one order of magnitude compared to that for the wild type enzyme. Catalytic efficiency of C115H MGL in the β-elimination reaction of S-alkylcysteines proved to be comparable to that for the wild type MGL [11]. Thus the replacement of Cys115 for histidine did not change catalytic properties of the enzyme in the β-elimination reaction. Obviously in the case of the γ-elimination reaction, the replacement did not disturb neither the stage of transaldimination nor the stage of Cα-proton abstraction (Scheme 1) which are common for the β- and γelimination reactions. In the spectrum of the C115H mutant enzyme complexed with methionine, the band belonging to the aminocrotonate is absent [11] while it is present in the analogous spectrum of the wild type MGL [16]. Hence the replacement interrupts the stage of methythiol elimination. The mutant form of P. putida MGL with the replacement of homologous cysteine 116 by histidine catalyzed the γ-elimination reaction of methionine with catalytic efficiency decreased for about four orders of magnitude compared to that of wild type enzyme. Meanwhile, our results [11] are not corresponding to the increased catalytic efficiency of the mutant form of P. putida MGL with the replacement of cysteine 116 in the β-elimination reaction [13,14]. The elimination of a γ-substituent needs an acid catalyst to proceed. A dual role was proposed for the active site tyrosine residue (Tyr113 of C. freundii MGL) conserved in the family of PLP-dependent enzymes of the cystathionine β-lyase subclass [4,25,26]. It was proposed that it acts as a base, abstracting a proton from an amino group of the incoming substrate to allow its nucleophilic attack on the C4′-atom of the internal aldimine [4,25]. Data on the pH-dependence of the γ-elimination reaction of methionine catalyzed by C. freundii MGL [27] and the spatial structures of MGL complexes with amino acids modeling Michaelis complexes [24] suggests that Tyr113 fulfills this role. This residue was proposed to be a general acid catalyst in the γ-elimination reaction 1126
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catalyzed by the enzymes of the cystathionine β-lyase subclass [4,25,26]. The replacement of Tyr114 of P. putida MGL by Phe gave evidence that the residue might perform acidic catalysis at the stage of methylthiol elimination [28]. The analogous mutant form of C. freundii with the replacement of Tyr113 by Phe catalyzed the γ-elimination of methionine with the catalytic efficiency reduced by four orders of magnitude as well (unpublished data). Comparison of the spatial structures of the C. freundii [15] and P. putida [29] MGLs, the structures of P. putida MGL C116H mutant form complexed with two substrates [14] and the structure of the external aldimine of C. freundii MGL with glycine [21] revealed the network of H-bonding contacts between conservative residues Tyr113, Cys115, Lys239, Asp240 (the numeration for C. freundii MGL) in MGL from different bacteria. The contacts become shorter on going from the structure of C. freundii MGL holoenzyme to the structure of the external aldimine with glycine [21]. This led us to propose that the proton abstracted from an amino group of a substrate could be stored at Cys115 or at Asp240 and then could be returned to Tyr113 to catalyze the stage of methylthiol elimination [8]. It was discussed above that the catalytic properties of the C115H mutant enzyme are evidence for which the mutant form catalyzes the stage of transaldimination, i.e. in the reactions of C115H enzyme with substrates of the γ- and β-elimination reactions, the Tyr113 hydroxylic group acts as a base. In the structure of the Michaelis complex, the nitrogen atom of the amino group of L-norleucine is 3.08 Å from the C4′ atom of the cofactor, thus making it possible to perform a nucleoрhilic attack on it. Spectral data on the C115H holoenzyme revealed that the main ionic form of the internal aldimine is a ketoenamine, i.e. the form with a positively charged aldimine nitrogen [11]. A nucleophility of the L-norleucine amino group may be provided by its closeness to the positive charge of the nitrogen atom of the ketoenamine. It is possible that the proton abstracted from the amino group of L-norleucine remains at the Tyr113 hydroxylic group as a short H-bond to the amino group of Lnorleucine holds it back. The closeness of the hydroxylic group of Tyr113 to the imidazole group of His115 creates favorable conditions for the formation of an Hbond between these groups. The pKa value of Tyr113 in the wild type enzyme was estimated at 7.1–7.2 [27], which is pretty close to the pKa of imidazole. Thus, even the possibility of formation of a low-barrier Hbond may be considered under certain circumstances. If such interactions take place at the stage of methylthiol elimination, Tyr113 should be unable to be a general acid catalyst because its hydroxylic proton is involved in the direct interaction with His115. These considerations may explain the absence of the γ-elimination reaction for C115H MGL. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2017.06.001.
[4]
[5]
[6]
[7]
[8]
[9]
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[16]
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[21]
Transparency Document [22]
The Transparency document associated with this article can be found, in online version. Acknowledgements
[23]
This work was supported by the Russian Science Foundation (project No. 15-14-00009). We thank Dr. Nicolai G. Faleev for critical reading of the manuscript.
[24]
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[25]
[26]
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