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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) 161e167
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Research paper
Crystal structure of the external aldimine of Citrobacter freundii methionine g-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of a- and b-protons of competitive inhibitors Svetlana V. Revtovich a,1, Nicolai G. Faleev b,1, Elena A. Morozova a, Natalya V. Anufrieva a, Alexey D. Nikulin c, Tatyana V. Demidkina a, * a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str. 32, Moscow 119991, Russia Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov Str. 28, Moscow 119991, Russia c Institute of Protein Research, Russian Academy of Sciences, Institutskaya Str. 4, 142290 Pushchino, Moscow Region, Russia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2013 Accepted 9 January 2014 Available online 24 January 2014
The three-dimensional structure of the external aldimine of Citrobacter freundii methionine g-lyase with competitive inhibitor glycine has been determined at 2.45 A resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Ca-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Ca-proton to C40 -atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Ca-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Ca-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Ca-atom of glycine to C40 -atom of the cofactor. The exchange of Ca-pro-(S)proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of b-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Pyridoxal 50 -phosphate dependent enzyme Methionine g-lyase Isotopic exchange of protons Concerted mechanism Stereoselectivity
1. Introduction Methionine g-lyase (MGL, EC 4.4.1.11) is a pyridoxal 50 -phosphate (PLP)-dependent enzyme that catalyzes the g-elimination reaction of methionine to give ketobutyric acid, methanethiol, and ammonia (Scheme 1). Besides physiological reaction the enzyme catalyzes g-replacement reactions of L-methionine and its analogs as well as b-elimination and b-replacement reactions of L-cysteine and S-substituted L-cysteines [1]. MGL has been found in a significant number of bacteria [2] including pathogenic bacteria Porphyromonas gingivalis [3], Clostridium sporogenes [4], Clostridium tetani [5]. It has been purified from two eukaryotic pathogens Entamoeba histolytica [6]
Abbreviations: MGL, methionine g-lyase; PLP, pyridoxal 50 -phosphate. * Corresponding author. Tel.: þ7 4991359858. E-mail address: [email protected] (T.V. Demidkina). 1 These authors contributed equally. 0300-9084/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2014.01.007
and Trichomonas vaginalis [7]. The enzyme is involved in the catabolism of methionine in Arabidopsis thaliana [8]. The absence of MGL in mammals makes it possible to consider the enzyme as a potential target for antimicrobial therapy. The toxicity of thiocarbonyl difluoride, the product of L-trifluoromethionine decomposition by MGL, may lead to suppression of growth of bacteria, which was demonstrated for T. vaginalis [9], P. gingivalis [10] and E. histolytica [11] in vitro and in vivo. The approaches to utilization of the enzyme as an effective antitumor drug are under intensive research [12]. The perspectives of using MGL to treat Parkinson disease, atherosclerosis, obesity, as well as its use in gerontology have been discussed [13]. Crystal structures of MGL holoenzymes of Pseudomonas putida (PDB: 2O7C) [14], Citrobacter freundii (PDB: 2RFV) [15] and T. vaginalis (PDB: 1E5F) have been solved. Spatial structures modeling Michaelis complexes of C. freundii MGL with substrates of g- and b-elimination reactions (PDB: 3JW9, 3JWA) and competitive inhibitor L-Nle (PDB: 3JWB) [16] and external aldimine with ammonia (PDB: 3MKJ) [17] have been determined. The structure of
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S.V. Revtovich et al. / Biochimie 101 (2014) 161e167 Table 1 Data collection and refinement statistics. Values in parentheses are for the highest resolution shell. Scheme 1.
T. vaginalis MGL complexed with non-specific irreversible inhibitor L-propargylglycine (PDB: 1E5E) and that of E. histolytica MGL complexed with methionine, as well as X-ray structures of putative intermediates of methionine decomposition by E. histolytica enzyme (PDB: 3AEO, 3AEN, 3AEM, 3AEL and 3AEP) have been solved. There are some data on substrate and reaction specificity of MGL from P. putida [1,18,19], T. vaginalis [7,20], E. histolytica [11] and C. freundii [21,22]. The role of the ionic state of a substrate in the mechanisms of g- and b-elimination reactions, catalyzed by C. freundii MGL, was examined [23]. However, the known data are not sufficient for a thorough understanding of mechanisms of various reactions catalyzed by the enzyme, which is quite necessary in view of the broad perspectives of its application in medicine. In this paper we present the crystal structure of the complex of C. freundii MGL with a competitive inhibitor glycine, modeling the structure of the external aldimine. It reveals some structural features responsible for the effective binding of substrates and inhibitors. Basing on this structure we made some mechanistic conclusions in order to explain the strict stereoselectivity of isotopic exchange of glycine Ca-protons and the lack of selectivity for the isotopic exchange of Cb-protons which is observed with amino acids containing longer side groups.
Space group Unit cell parameters ( A) Wavelength ( A) Resolution ( A) Completeness (%) Redundancy Rmerge (%) Disordered protein residues No. of non-H protein atoms No. of water atoms No. of unique reflections R/Rfree Mean temperature factor B ( A2) R.m.s. deviation from ideal values Bond lengths ( A) Bond angles ( ) Chirality angles ( ) Planar angles ( ) Ramachandran plot Favored region (%) Allowed region (%) Outlier region (%)
I222 a ¼ 56.29, b ¼ 123.05, c ¼ 126.75 1.54 35e2.45 (2.51e2.45) 98.3 (99.4) 5.04 (3.95) 9.7 (45) 1, 398 2999 211 15,499 0.224/0.337 (0.240/0.324) 33.32 0.013 1.730 0.118 0.008 94.4 4.6 1.0
structure has been submitted to the Protein Data Bank with PDB: 4HF8.
3. Results and discussion 3.1. Overall structure of the complex
2. Experimental 2.1. Crystallization and data collection MGL from C. freundii was isolated and purified as described by Manukhov et al., 2005 [21]. Crystals of MGL were obtained using the same conditions as described by Mamaeva et al., 2005 [24], but without the presence of ammonium sulfate in the solutions. Rhombic-shaped crystals appeared after a week and attained dimensions of 0.3 mm within two weeks. The complex of MGL with glycine was obtained by soaking of holoenzyme crystals in a cryoprotective mother liquid solution, containing glycine (20 mM), during 20 min. Glycine was added to the solution gradually, at first to reach a concentration of 5 mM, which was further increased in two steps up to 20 mM. Stepwise addition was necessary to avoid a cracking of crystals observed when glycine was added in one step. Data from a single crystal were collected at Institute of Protein Research (Pushchino, Russia) using X8 Proteum (Bruker-AXS) instrument with a Platinum135 CCD detector and processed by the Proteum software. The detailed data collecting statistics are shown in Table 1. 2.2. Structure determination and refinement The structure was solved by molecular replacement using the structure of C. freundii MGL (PDB: 1Y4I) by rigid body procedure, implemented in CCP4 software suite [25]. The model was improved using manual rebuilding with a COOT [26] and maximum likelihood refinement using REFMAC5 [27]. Flexible loops of the protein and water molecules were removed from the initial model to exclude model bias during the first round of refinement. The final model, refined to 2.45 A, incorporated 2999 nonhydrogen atoms. The model included the external aldimine, PEG and 211 water molecules converged to Rwork of 22.4% and Rfree of 33.7% for the data between 35.0 A and 2.45 A (Table 1). The
The fold of polypeptide chain of C. freundii MGL (PDB: 1Y4I) [24] has characteristic features of PLP-dependent enzymes type I fold [28], subclass of cystathionine b-lyase [29]. Tetrameric molecule should be considered as composed of two catalytic dimers related by a twofold axis. Each dimer contains two active sites formed by amino acid residues from both subunits. Each monomer consists of three domains: N-terminal domain, central PLP-binding domain, and C-terminal domain. Binding of glycine did not lead to any significant influence on overall fold of MGL polypeptide chain as compared to that of holoenzyme. The marked structural differences are concerned with the positions of mobile parts of N-terminal and C-terminal domains. The flexible part of N-terminal domain (residues 47e63) includes several amino acids of helix a2, irregular region and the turn of Ca-chain until helix a3 of PLP-binding domain. The mobility of this part of the polypeptide chain is so evident, that in some earlier determined MGL structures (PDB: 2RFV and 3MKJ) it is not possible to localize all amino acid residues. Nevertheless, amino acids of this region are of importance due to taking part in formation of active site hydrophobic pocket (Phe49, Leu57 and Leu61), as well as enzyme active site (Tyr58 and Arg60). Despite difference between positions of Tyr58 and Arg60 in two holoenzyme structures (PDB: 2RFV and 1Y4I) and in the structure of complex with glycine, these two residues have the same arrangement in structures of MGL complexes with substrates, phosphinic analog of methionine (PDB: 3JW9) and S-ethyl-L-cysteine (PDB: 3JWA) and inhibitors, norleucine (PDB: 3JWB) and glycine. We suppose that both residues occupy the optimal position to provide the enzymatic reaction in the observed structure. Side chain of Arg60* is located along PLP “handle” toward the Tyr113. In this orientation it forms two H-bonds with PLP “handle” Nε and Nh atoms and one H-bond between Nh atom and hydroxyl group of Tyr113. Tyr58* is located at the other side of PLP “handle” than Arg60*, and it forms a short H-bond between hydroxyl group and O1P atom of PLP (Fig. 1).
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The flexible part of C-terminal domain (residues 353e368) includes a14 helix located at the interface between PLP-binding and C-terminal domains [15]. Ca polypeptide chain of this region does not change its position but in the discussed structure some reducing of electron density is observed and the protein tracing has been embarrassing. Average B-factor of this region increases almost twice as compared with average B-factor of residues 353e368 of holoenzyme (PBD: 2RFV) and proved to be about 84 Å2. This region is well ordered in the holoenzyme structures but is poorly visible in Michaelis complexes of MGL with substrates and inhibitors [16]. It seems that this large segment of the C-terminal domain makes a part of a shutter, covering the entrance to the substrate binding pocket of MGL, whereas the flexible part of N-terminal domain forms another one. An additional short flexible region is a turn between b4-sheet and a7-helix (residues 160e163). Its flexibility leads to a positioning of the side chain of Asn160 at H-bond distances to 30 -hydroxyl group of the cofactor, side chain of substrate-binding Arg374, and carboxyl group of glycine. A net of H-bonds formed by the side chain of Asn160 at the active site demonstrates that movement of the short region (residues 160e163) is important for providing effective substrate binding and the abstraction of Caproton. This residue is conserved in primary structures of MGL from different microorganisms [30] and it is structurally invariant in spatial structures of a number of PLP-dependent enzymes of type I fold. Its specific role in catalysis varies depending on an enzyme. Similarly to non-covalent complexes of MGL with substrates and inhibitors [16], there is a 180 switch of main chain C]O group between residues Val338 and Ser339 relative to its position in the holoenzyme. The position of the carbonyl group in the holoenzyme does not enable binding of the substrates and inhibitors (Fig. 2). Consequently, this turn is obligatory to accept an amino acid. Analogous turn was observed in X-ray structures of covalent (T. vaginalis MGL, PDB: 1E5E) and non-covalent (Homo sapiens cystathionine g-lyase, PDB: 3COG) complexes of two enzymes belonging to the cystathionine b-lyase subclass with Lpropargylglycine. 3.2. Active site All the reactions of amino acids metabolism catalyzed by PLPdependent enzymes require a formation of an external aldimine by the transaldimination reaction between Schiff base, formed by the side chain of active site Lys with aldehyde group of PLP, and amino group of a substrate (Fig. 3).
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Fig. 2. Superposition of C]O group between Val338 and Ser339 in the holoenzyme (PDB: 2RFV, blue) and in the external aldimine of MGL with glycine. “Forbidden” distance between a carboxylate oxygen of glycine and C]O group of polypeptide chain is indicated by red dashed line.
Spectral measurements of MGL interaction with amino acids in a single crystal demonstrated that the external aldimine predominates in the equilibrium mixture of the enzyme with glycine [17]. In the X-ray structure it is clearly seen that glycine is covalently bound to the cofactor (Fig. 4). Its binding is provided by the carboxyl group of the glycine which is involved in salt-bridge and H-bond interactions with the active side residues. It makes a characteristic “bracket” with the side chain atoms of Arg374 and H-bonds with main chain nitrogen atom of Ser339 and amide nitrogen of Asn160 (Fig. 5). The released side chain of Lys210 forms H-bond with the Ser207 hydroxyl group. After the stage of external aldimine formation the majority of reactions catalyzed by PLP-dependent enzymes demand the abstraction of Ca-proton from the external aldimine and usually this stage is catalyzed by the active site lysine residue released upon the external aldimine formation. According to the spectral data the external aldimine with glycine exists in the ionic form of ketoenamine with positively charged aldimine nitrogen both in the solution [22] and in the crystal [17]. Side amino group of Lys210 should be uncharged because it is at 3.4 A distance from positive aldimine nitrogen. It is at 3.6 A distance from Ca-atom of the
Fig. 1. Stereo view of the superposition of Arg60* and Tyr58* residues inside of active site in two holoenzymes (PDB: 2RFV, cyan, and PDB: 1Y4I, red) and in the external aldimine of MGL with glycine (gray). H-bonds are indicated by dashed lines.
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Fig. 3. Initial intermediates of g-elimination reaction.
Fig. 4. Stereo representation of the external aldimine with the final 2Fo Fc map contoured at the 1.0 s level.
external aldimine. Consequently, it may act as an effective base at the stage of Ca-proton abstraction from the external aldimine with quinonoid intermediate formation. The H-bond between the amide group of Asn160 and 30 -oxygen of PLP should increase the acidity of Ca-proton by withdrawing of electrons from 30 -oxygen atom. The next stage along the g-elimination reaction pathway is the protonation of C40 -atom of PLP with ketimine formation (Fig. 3).
This intermediate was shown to be obligatory for PLP-dependent gelimination reaction to proceed [31]. The side chain of Lys210 is at a distance of 2.6 A from C40 -atom and thus has a favorable position to perform the transfer of the abstracted Ca-proton to C40 -atom of the cofactor to form ketimine. Alternatively one may assume that there might be a concerted mechanism of ketimine formation through a cyclic six-member transition state with involvement of nitrogen
Fig. 5. Stereo view of the external aldimine active site. The superposition of the holoenzyme (PDB: 2RFV) residues is shown in azure. H-bonds are indicated by green dashed lines.
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atom and a proton of the amino group of Lys210 (Fig. 6). The role of the active site Lys residue which could move “like a liana” to perform Ca- and Cb-protons abstractions and 1e3 prototropic shift in the courses of elimination and replacement reactions at g-carbon atom of cystathionine catalyzed by cystathionine g-synthase was proposed by Clausen et al., 1998 [32]. In the external aldimine there are tilts of PLP ring around the C5eC2 axis and Tyr113 ring to the solvent as compared to their positions in the holoenzyme (17 and 28 respectively). As a result hydroxyl group of Tyr113 approaches at H-bond distances (3.2 A) to sulfur atom of Cys115 and to side chain nitrogen (2.6 A) of Arg60*. Phenolic oxygen of Tyr113 is at 4.1 A distance from positively charged nitrogen of aldimine bond. Probably side chain of Tyr113 exists predominantly as phenolate anion. 3.3. The structure of external aldimine and the mechanism of isotopic exchange of glycine protons MGL catalyzes isotopic exchange of both the pro-(R)- and pro(S)-protons of glycine in 2H2O [33,34]. According to the stereoelectronic hypothesis of Dunathan [35] the orthogonal orientation of the Ca-proton of an external aldimine with respect to the cofactor plane is obligatory for its labilization and respectively for the possibility of the isotopic exchange. The other condition for the isotopic exchange is the presence of a suitable catalytic group in close proximity. In the structure under consideration the pro-(R)proton of glycine has the required orientation and is located on the re-side of the cofactor plane at a distance of 2.8 A from the amino group of Lys210, the latter being in the form of a free base. On the other side of the cofactor plane the pro-(S)-proton is located practically at the same distance from the hydroxyl group of Tyr113 (Fig. 7). A similar construction with Lys and Tyr residues at different sides of the PLP plane is operative in the case of alanine racemase [36] and side racemization reaction catalyzed by cystalysin [37]. If side chain of Tyr113 is in the acidic form, the observed position of the catalytic groups looks very favorable for the realization of the mechanism of the isotopic exchange with the inversion of configuration at Ca-atom. This mechanism is concerted and implies the abstraction of pro-(R)-proton by the amino group of Lys210, and simultaneous deuteration of Ca-atom under the action of Tyr113 from the opposite side. If the inversion mechanism were operative in the considered case the rates of the exchange would be practically the same for both protons. Surprisingly, in fact the exchange is highly stereoselective: pro-(R)-proton reacts by a factor of 1440 faster than does pro-(S)-proton [33]. We believe that in the considered structure Tyr113 exists in the basic form which is stabilized by interaction with positively charged Arg60* from neighboring subunit, by positive charge of aldimine nitrogen, and probably with Cys115 located nearby. In this case the two-
Fig. 7. Locations of two possible bases for pro-(S)- and pro-(R)-protons exchange.
enantiotropic protons should be exchanged with retention of configuration under the action of two different basic groups. Due to the mentioned above stabilizing interactions the basicity of Tyr113 is strongly decreased and, consequently, the isotopic exchange of the pro-(S)-proton is retarded. As regards the fast exchange of pro(R)-proton, it may become even more favorable because another mechanism of exchange is realized. This mechanism also is accompanied by retention of configuration at Ca-atom and implies a concerted transfer of the pro-(R)-proton from Ca-atom to C40 atom (Fig. 7) which may be fast and reversible. Such mechanism may be realized with participation of amino group, bearing two protons (two deuterons in 2H2O), but is not possible with dissociated hydroxyl group of Tyr113.
Fig. 6. Possible concerted mechanisms of reversible ketimine formation and pro-(R)-Ca-proton exchange.
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The suggested mechanism of the isotopic exchange of glycine protons implies participation of two basic groups (Lys210 and Tyr113) located at different sides of the cofactor plane in very close proximity to the Ca-atom of glycine. It is known that interacting with amino acids containing longer side chains MGL catalyzes the exchange of not only a-protons, but of b-protons too. Interestingly, both b-protons are exchanged at practically the same rates [33,34]. For the exchange of b-protons deprotonation of Ca-atom and subsequent protonation of C40 -atom of the cofactor are obligatory [31]. Most probably the both processes are effected by Lys210 residue which after all remains in the basic form and participates in the following exchange of b-protons. Taking into account that both bprotons are exchanged at the same rates the most reasonable mechanism is the mechanism with inversion of configuration at Cbatom. Besides Lys residue it requires also the participation of an additional catalytic group, existing in acidic form and placed at the opposite side of the cofactor plane. The most probable candidate on this role is Tyr113 residue, especially in view of the fact [19] that replacement of Tyr114 in MGL from P. putida (which is homologous to Tyr113 in the studied enzyme) for Phe makes the enzyme capable to exchange only one of b-protons. These considerations allow us to conclude that the protonation state of Tyr113 changes during the stages where the direct participation of this residue is not needed. It can be changed in the course of ketimine formation which is necessary for the labilization of b-protons. One may assume that conformational changes of the protein associated with the binding of different amino acids may be accompanied by changes in ionic state of active site groups. In this case binding of glycine may be followed by principally different transfers. We may conclude that in the case of MGL catalytic events proceed at both sides of the cofactor plane, and, consequently, the principle of “one sided reactivity” [34] in the chemistry of PLP-dependent enzymes may by applied in more limited number of cases than it was supposed previously. Acknowledgments This work was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Researches (N 11-04-01535-a, N 14-04-00349-a) and the grant of the President of the Russian Federation for state support of the leading scientific school (N 2046.2014.4). References [1] H. Tanaka, N. Esaki, K. Soda, A versatile bacterial enzyme: L-methionine glyase, Enzyme Microb. Technol. 7 (1985) 530e537. [2] A.S. El-Sayed, Microbial L-methioninase: production, molecular characterization, and therapeutic applications, Appl. Microbiol. Biotechnol. 86 (2010) 445e467. [3] M. Yoshimura, Y. Nakano, Y. Yamashita, T. Oho, T. Saito, T. Koga, Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis, Infect. Immun. 68 (2000) 6912e6916. [4] W. Kreis, C. Hession, Isolation and purification of L-methionine-a-deamino-gmercaptomethane-lyase (L-methioninase) from Clostridium sporogenes, Cancer Res. 33 (1973) 1862e1865. [5] S.V. Revtovich, E.A. Morozova, N.V. Anufrieva, M.I. Kotlov, Y.F. Belyi, T.V. Demidkina, Identification of methionine g-lyase in genomes of some pathogenic bacteria, Dokl. Biochem. Biophys. 445 (2012) 187e193. [6] M. Tokoro, T. Asai, S. Kobayashi, T. Takeuchi, T. Nozaki, Identification and characterization of two isoenzymes of methionine g-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways, J. Biol. Chem. 278 (2003) 42717e42727. [7] B. Lockwood, G. Coombs, Purification and characterization of methionine glyase from Trichomonas vaginalis, Biochem. J. 279 (1991) 675e682. [8] F. Rebeille, S. Jabrin, R. Bligny, K. Loizeau, B. Gambonnet, V. Van Wilder, R. Douce, S. Ravanel, Methionine catabolism in Arabidopsis cells is initiated by a g-cleavage process and leads to S-methylcysteine and isoleucine syntheses, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15687e15692.
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Lindqvist, Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal 50 -phosphate-dependent enzymes, J. Mol. Biol. 291 (1999) 857e876. [30] I.V. Manukhov, D.V. Mamaeva, E.A. Morozova, S.M. Rastorguev, N.G. Faleev, T.V. Demidkina, G.B. Zavilgelsky, L-Methionine g-lyase from Citrobacter freundii cloning of the gene and kinetic parameters of the enzyme, Biochemistry (Moscow) 71 (2006) 361e369. [31] P. Brzovic, E.L. Holbrook, R.C. Greene, M.F. Dunn, Reaction mechanism of Escherichia coli cystathionine g-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent g-elimination and g-replacement reactions, Biochemistry 29 (1990) 442e451.
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Research paper
Crystal structure of the external aldimine of Citrobacter freundii methionine g-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of a- and b-protons of competitive inhibitors Svetlana V. Revtovich a,1, Nicolai G. Faleev b,1, Elena A. Morozova a, Natalya V. Anufrieva a, Alexey D. Nikulin c, Tatyana V. Demidkina a, * a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str. 32, Moscow 119991, Russia Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov Str. 28, Moscow 119991, Russia c Institute of Protein Research, Russian Academy of Sciences, Institutskaya Str. 4, 142290 Pushchino, Moscow Region, Russia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2013 Accepted 9 January 2014 Available online 24 January 2014
The three-dimensional structure of the external aldimine of Citrobacter freundii methionine g-lyase with competitive inhibitor glycine has been determined at 2.45 A resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Ca-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Ca-proton to C40 -atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Ca-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Ca-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Ca-atom of glycine to C40 -atom of the cofactor. The exchange of Ca-pro-(S)proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of b-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Pyridoxal 50 -phosphate dependent enzyme Methionine g-lyase Isotopic exchange of protons Concerted mechanism Stereoselectivity
1. Introduction Methionine g-lyase (MGL, EC 4.4.1.11) is a pyridoxal 50 -phosphate (PLP)-dependent enzyme that catalyzes the g-elimination reaction of methionine to give ketobutyric acid, methanethiol, and ammonia (Scheme 1). Besides physiological reaction the enzyme catalyzes g-replacement reactions of L-methionine and its analogs as well as b-elimination and b-replacement reactions of L-cysteine and S-substituted L-cysteines [1]. MGL has been found in a significant number of bacteria [2] including pathogenic bacteria Porphyromonas gingivalis [3], Clostridium sporogenes [4], Clostridium tetani [5]. It has been purified from two eukaryotic pathogens Entamoeba histolytica [6]
Abbreviations: MGL, methionine g-lyase; PLP, pyridoxal 50 -phosphate. * Corresponding author. Tel.: þ7 4991359858. E-mail address: [email protected] (T.V. Demidkina). 1 These authors contributed equally. 0300-9084/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2014.01.007
and Trichomonas vaginalis [7]. The enzyme is involved in the catabolism of methionine in Arabidopsis thaliana [8]. The absence of MGL in mammals makes it possible to consider the enzyme as a potential target for antimicrobial therapy. The toxicity of thiocarbonyl difluoride, the product of L-trifluoromethionine decomposition by MGL, may lead to suppression of growth of bacteria, which was demonstrated for T. vaginalis [9], P. gingivalis [10] and E. histolytica [11] in vitro and in vivo. The approaches to utilization of the enzyme as an effective antitumor drug are under intensive research [12]. The perspectives of using MGL to treat Parkinson disease, atherosclerosis, obesity, as well as its use in gerontology have been discussed [13]. Crystal structures of MGL holoenzymes of Pseudomonas putida (PDB: 2O7C) [14], Citrobacter freundii (PDB: 2RFV) [15] and T. vaginalis (PDB: 1E5F) have been solved. Spatial structures modeling Michaelis complexes of C. freundii MGL with substrates of g- and b-elimination reactions (PDB: 3JW9, 3JWA) and competitive inhibitor L-Nle (PDB: 3JWB) [16] and external aldimine with ammonia (PDB: 3MKJ) [17] have been determined. The structure of
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S.V. Revtovich et al. / Biochimie 101 (2014) 161e167 Table 1 Data collection and refinement statistics. Values in parentheses are for the highest resolution shell. Scheme 1.
T. vaginalis MGL complexed with non-specific irreversible inhibitor L-propargylglycine (PDB: 1E5E) and that of E. histolytica MGL complexed with methionine, as well as X-ray structures of putative intermediates of methionine decomposition by E. histolytica enzyme (PDB: 3AEO, 3AEN, 3AEM, 3AEL and 3AEP) have been solved. There are some data on substrate and reaction specificity of MGL from P. putida [1,18,19], T. vaginalis [7,20], E. histolytica [11] and C. freundii [21,22]. The role of the ionic state of a substrate in the mechanisms of g- and b-elimination reactions, catalyzed by C. freundii MGL, was examined [23]. However, the known data are not sufficient for a thorough understanding of mechanisms of various reactions catalyzed by the enzyme, which is quite necessary in view of the broad perspectives of its application in medicine. In this paper we present the crystal structure of the complex of C. freundii MGL with a competitive inhibitor glycine, modeling the structure of the external aldimine. It reveals some structural features responsible for the effective binding of substrates and inhibitors. Basing on this structure we made some mechanistic conclusions in order to explain the strict stereoselectivity of isotopic exchange of glycine Ca-protons and the lack of selectivity for the isotopic exchange of Cb-protons which is observed with amino acids containing longer side groups.
Space group Unit cell parameters ( A) Wavelength ( A) Resolution ( A) Completeness (%) Redundancy Rmerge (%) Disordered protein residues No. of non-H protein atoms No. of water atoms No. of unique reflections R/Rfree Mean temperature factor B ( A2) R.m.s. deviation from ideal values Bond lengths ( A) Bond angles ( ) Chirality angles ( ) Planar angles ( ) Ramachandran plot Favored region (%) Allowed region (%) Outlier region (%)
I222 a ¼ 56.29, b ¼ 123.05, c ¼ 126.75 1.54 35e2.45 (2.51e2.45) 98.3 (99.4) 5.04 (3.95) 9.7 (45) 1, 398 2999 211 15,499 0.224/0.337 (0.240/0.324) 33.32 0.013 1.730 0.118 0.008 94.4 4.6 1.0
structure has been submitted to the Protein Data Bank with PDB: 4HF8.
3. Results and discussion 3.1. Overall structure of the complex
2. Experimental 2.1. Crystallization and data collection MGL from C. freundii was isolated and purified as described by Manukhov et al., 2005 [21]. Crystals of MGL were obtained using the same conditions as described by Mamaeva et al., 2005 [24], but without the presence of ammonium sulfate in the solutions. Rhombic-shaped crystals appeared after a week and attained dimensions of 0.3 mm within two weeks. The complex of MGL with glycine was obtained by soaking of holoenzyme crystals in a cryoprotective mother liquid solution, containing glycine (20 mM), during 20 min. Glycine was added to the solution gradually, at first to reach a concentration of 5 mM, which was further increased in two steps up to 20 mM. Stepwise addition was necessary to avoid a cracking of crystals observed when glycine was added in one step. Data from a single crystal were collected at Institute of Protein Research (Pushchino, Russia) using X8 Proteum (Bruker-AXS) instrument with a Platinum135 CCD detector and processed by the Proteum software. The detailed data collecting statistics are shown in Table 1. 2.2. Structure determination and refinement The structure was solved by molecular replacement using the structure of C. freundii MGL (PDB: 1Y4I) by rigid body procedure, implemented in CCP4 software suite [25]. The model was improved using manual rebuilding with a COOT [26] and maximum likelihood refinement using REFMAC5 [27]. Flexible loops of the protein and water molecules were removed from the initial model to exclude model bias during the first round of refinement. The final model, refined to 2.45 A, incorporated 2999 nonhydrogen atoms. The model included the external aldimine, PEG and 211 water molecules converged to Rwork of 22.4% and Rfree of 33.7% for the data between 35.0 A and 2.45 A (Table 1). The
The fold of polypeptide chain of C. freundii MGL (PDB: 1Y4I) [24] has characteristic features of PLP-dependent enzymes type I fold [28], subclass of cystathionine b-lyase [29]. Tetrameric molecule should be considered as composed of two catalytic dimers related by a twofold axis. Each dimer contains two active sites formed by amino acid residues from both subunits. Each monomer consists of three domains: N-terminal domain, central PLP-binding domain, and C-terminal domain. Binding of glycine did not lead to any significant influence on overall fold of MGL polypeptide chain as compared to that of holoenzyme. The marked structural differences are concerned with the positions of mobile parts of N-terminal and C-terminal domains. The flexible part of N-terminal domain (residues 47e63) includes several amino acids of helix a2, irregular region and the turn of Ca-chain until helix a3 of PLP-binding domain. The mobility of this part of the polypeptide chain is so evident, that in some earlier determined MGL structures (PDB: 2RFV and 3MKJ) it is not possible to localize all amino acid residues. Nevertheless, amino acids of this region are of importance due to taking part in formation of active site hydrophobic pocket (Phe49, Leu57 and Leu61), as well as enzyme active site (Tyr58 and Arg60). Despite difference between positions of Tyr58 and Arg60 in two holoenzyme structures (PDB: 2RFV and 1Y4I) and in the structure of complex with glycine, these two residues have the same arrangement in structures of MGL complexes with substrates, phosphinic analog of methionine (PDB: 3JW9) and S-ethyl-L-cysteine (PDB: 3JWA) and inhibitors, norleucine (PDB: 3JWB) and glycine. We suppose that both residues occupy the optimal position to provide the enzymatic reaction in the observed structure. Side chain of Arg60* is located along PLP “handle” toward the Tyr113. In this orientation it forms two H-bonds with PLP “handle” Nε and Nh atoms and one H-bond between Nh atom and hydroxyl group of Tyr113. Tyr58* is located at the other side of PLP “handle” than Arg60*, and it forms a short H-bond between hydroxyl group and O1P atom of PLP (Fig. 1).
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The flexible part of C-terminal domain (residues 353e368) includes a14 helix located at the interface between PLP-binding and C-terminal domains [15]. Ca polypeptide chain of this region does not change its position but in the discussed structure some reducing of electron density is observed and the protein tracing has been embarrassing. Average B-factor of this region increases almost twice as compared with average B-factor of residues 353e368 of holoenzyme (PBD: 2RFV) and proved to be about 84 Å2. This region is well ordered in the holoenzyme structures but is poorly visible in Michaelis complexes of MGL with substrates and inhibitors [16]. It seems that this large segment of the C-terminal domain makes a part of a shutter, covering the entrance to the substrate binding pocket of MGL, whereas the flexible part of N-terminal domain forms another one. An additional short flexible region is a turn between b4-sheet and a7-helix (residues 160e163). Its flexibility leads to a positioning of the side chain of Asn160 at H-bond distances to 30 -hydroxyl group of the cofactor, side chain of substrate-binding Arg374, and carboxyl group of glycine. A net of H-bonds formed by the side chain of Asn160 at the active site demonstrates that movement of the short region (residues 160e163) is important for providing effective substrate binding and the abstraction of Caproton. This residue is conserved in primary structures of MGL from different microorganisms [30] and it is structurally invariant in spatial structures of a number of PLP-dependent enzymes of type I fold. Its specific role in catalysis varies depending on an enzyme. Similarly to non-covalent complexes of MGL with substrates and inhibitors [16], there is a 180 switch of main chain C]O group between residues Val338 and Ser339 relative to its position in the holoenzyme. The position of the carbonyl group in the holoenzyme does not enable binding of the substrates and inhibitors (Fig. 2). Consequently, this turn is obligatory to accept an amino acid. Analogous turn was observed in X-ray structures of covalent (T. vaginalis MGL, PDB: 1E5E) and non-covalent (Homo sapiens cystathionine g-lyase, PDB: 3COG) complexes of two enzymes belonging to the cystathionine b-lyase subclass with Lpropargylglycine. 3.2. Active site All the reactions of amino acids metabolism catalyzed by PLPdependent enzymes require a formation of an external aldimine by the transaldimination reaction between Schiff base, formed by the side chain of active site Lys with aldehyde group of PLP, and amino group of a substrate (Fig. 3).
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Fig. 2. Superposition of C]O group between Val338 and Ser339 in the holoenzyme (PDB: 2RFV, blue) and in the external aldimine of MGL with glycine. “Forbidden” distance between a carboxylate oxygen of glycine and C]O group of polypeptide chain is indicated by red dashed line.
Spectral measurements of MGL interaction with amino acids in a single crystal demonstrated that the external aldimine predominates in the equilibrium mixture of the enzyme with glycine [17]. In the X-ray structure it is clearly seen that glycine is covalently bound to the cofactor (Fig. 4). Its binding is provided by the carboxyl group of the glycine which is involved in salt-bridge and H-bond interactions with the active side residues. It makes a characteristic “bracket” with the side chain atoms of Arg374 and H-bonds with main chain nitrogen atom of Ser339 and amide nitrogen of Asn160 (Fig. 5). The released side chain of Lys210 forms H-bond with the Ser207 hydroxyl group. After the stage of external aldimine formation the majority of reactions catalyzed by PLP-dependent enzymes demand the abstraction of Ca-proton from the external aldimine and usually this stage is catalyzed by the active site lysine residue released upon the external aldimine formation. According to the spectral data the external aldimine with glycine exists in the ionic form of ketoenamine with positively charged aldimine nitrogen both in the solution [22] and in the crystal [17]. Side amino group of Lys210 should be uncharged because it is at 3.4 A distance from positive aldimine nitrogen. It is at 3.6 A distance from Ca-atom of the
Fig. 1. Stereo view of the superposition of Arg60* and Tyr58* residues inside of active site in two holoenzymes (PDB: 2RFV, cyan, and PDB: 1Y4I, red) and in the external aldimine of MGL with glycine (gray). H-bonds are indicated by dashed lines.
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Fig. 3. Initial intermediates of g-elimination reaction.
Fig. 4. Stereo representation of the external aldimine with the final 2Fo Fc map contoured at the 1.0 s level.
external aldimine. Consequently, it may act as an effective base at the stage of Ca-proton abstraction from the external aldimine with quinonoid intermediate formation. The H-bond between the amide group of Asn160 and 30 -oxygen of PLP should increase the acidity of Ca-proton by withdrawing of electrons from 30 -oxygen atom. The next stage along the g-elimination reaction pathway is the protonation of C40 -atom of PLP with ketimine formation (Fig. 3).
This intermediate was shown to be obligatory for PLP-dependent gelimination reaction to proceed [31]. The side chain of Lys210 is at a distance of 2.6 A from C40 -atom and thus has a favorable position to perform the transfer of the abstracted Ca-proton to C40 -atom of the cofactor to form ketimine. Alternatively one may assume that there might be a concerted mechanism of ketimine formation through a cyclic six-member transition state with involvement of nitrogen
Fig. 5. Stereo view of the external aldimine active site. The superposition of the holoenzyme (PDB: 2RFV) residues is shown in azure. H-bonds are indicated by green dashed lines.
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165
atom and a proton of the amino group of Lys210 (Fig. 6). The role of the active site Lys residue which could move “like a liana” to perform Ca- and Cb-protons abstractions and 1e3 prototropic shift in the courses of elimination and replacement reactions at g-carbon atom of cystathionine catalyzed by cystathionine g-synthase was proposed by Clausen et al., 1998 [32]. In the external aldimine there are tilts of PLP ring around the C5eC2 axis and Tyr113 ring to the solvent as compared to their positions in the holoenzyme (17 and 28 respectively). As a result hydroxyl group of Tyr113 approaches at H-bond distances (3.2 A) to sulfur atom of Cys115 and to side chain nitrogen (2.6 A) of Arg60*. Phenolic oxygen of Tyr113 is at 4.1 A distance from positively charged nitrogen of aldimine bond. Probably side chain of Tyr113 exists predominantly as phenolate anion. 3.3. The structure of external aldimine and the mechanism of isotopic exchange of glycine protons MGL catalyzes isotopic exchange of both the pro-(R)- and pro(S)-protons of glycine in 2H2O [33,34]. According to the stereoelectronic hypothesis of Dunathan [35] the orthogonal orientation of the Ca-proton of an external aldimine with respect to the cofactor plane is obligatory for its labilization and respectively for the possibility of the isotopic exchange. The other condition for the isotopic exchange is the presence of a suitable catalytic group in close proximity. In the structure under consideration the pro-(R)proton of glycine has the required orientation and is located on the re-side of the cofactor plane at a distance of 2.8 A from the amino group of Lys210, the latter being in the form of a free base. On the other side of the cofactor plane the pro-(S)-proton is located practically at the same distance from the hydroxyl group of Tyr113 (Fig. 7). A similar construction with Lys and Tyr residues at different sides of the PLP plane is operative in the case of alanine racemase [36] and side racemization reaction catalyzed by cystalysin [37]. If side chain of Tyr113 is in the acidic form, the observed position of the catalytic groups looks very favorable for the realization of the mechanism of the isotopic exchange with the inversion of configuration at Ca-atom. This mechanism is concerted and implies the abstraction of pro-(R)-proton by the amino group of Lys210, and simultaneous deuteration of Ca-atom under the action of Tyr113 from the opposite side. If the inversion mechanism were operative in the considered case the rates of the exchange would be practically the same for both protons. Surprisingly, in fact the exchange is highly stereoselective: pro-(R)-proton reacts by a factor of 1440 faster than does pro-(S)-proton [33]. We believe that in the considered structure Tyr113 exists in the basic form which is stabilized by interaction with positively charged Arg60* from neighboring subunit, by positive charge of aldimine nitrogen, and probably with Cys115 located nearby. In this case the two-
Fig. 7. Locations of two possible bases for pro-(S)- and pro-(R)-protons exchange.
enantiotropic protons should be exchanged with retention of configuration under the action of two different basic groups. Due to the mentioned above stabilizing interactions the basicity of Tyr113 is strongly decreased and, consequently, the isotopic exchange of the pro-(S)-proton is retarded. As regards the fast exchange of pro(R)-proton, it may become even more favorable because another mechanism of exchange is realized. This mechanism also is accompanied by retention of configuration at Ca-atom and implies a concerted transfer of the pro-(R)-proton from Ca-atom to C40 atom (Fig. 7) which may be fast and reversible. Such mechanism may be realized with participation of amino group, bearing two protons (two deuterons in 2H2O), but is not possible with dissociated hydroxyl group of Tyr113.
Fig. 6. Possible concerted mechanisms of reversible ketimine formation and pro-(R)-Ca-proton exchange.
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The suggested mechanism of the isotopic exchange of glycine protons implies participation of two basic groups (Lys210 and Tyr113) located at different sides of the cofactor plane in very close proximity to the Ca-atom of glycine. It is known that interacting with amino acids containing longer side chains MGL catalyzes the exchange of not only a-protons, but of b-protons too. Interestingly, both b-protons are exchanged at practically the same rates [33,34]. For the exchange of b-protons deprotonation of Ca-atom and subsequent protonation of C40 -atom of the cofactor are obligatory [31]. Most probably the both processes are effected by Lys210 residue which after all remains in the basic form and participates in the following exchange of b-protons. Taking into account that both bprotons are exchanged at the same rates the most reasonable mechanism is the mechanism with inversion of configuration at Cbatom. Besides Lys residue it requires also the participation of an additional catalytic group, existing in acidic form and placed at the opposite side of the cofactor plane. The most probable candidate on this role is Tyr113 residue, especially in view of the fact [19] that replacement of Tyr114 in MGL from P. putida (which is homologous to Tyr113 in the studied enzyme) for Phe makes the enzyme capable to exchange only one of b-protons. These considerations allow us to conclude that the protonation state of Tyr113 changes during the stages where the direct participation of this residue is not needed. It can be changed in the course of ketimine formation which is necessary for the labilization of b-protons. One may assume that conformational changes of the protein associated with the binding of different amino acids may be accompanied by changes in ionic state of active site groups. In this case binding of glycine may be followed by principally different transfers. We may conclude that in the case of MGL catalytic events proceed at both sides of the cofactor plane, and, consequently, the principle of “one sided reactivity” [34] in the chemistry of PLP-dependent enzymes may by applied in more limited number of cases than it was supposed previously. Acknowledgments This work was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Researches (N 11-04-01535-a, N 14-04-00349-a) and the grant of the President of the Russian Federation for state support of the leading scientific school (N 2046.2014.4). References [1] H. Tanaka, N. Esaki, K. Soda, A versatile bacterial enzyme: L-methionine glyase, Enzyme Microb. Technol. 7 (1985) 530e537. [2] A.S. El-Sayed, Microbial L-methioninase: production, molecular characterization, and therapeutic applications, Appl. Microbiol. Biotechnol. 86 (2010) 445e467. [3] M. Yoshimura, Y. Nakano, Y. Yamashita, T. Oho, T. Saito, T. Koga, Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis, Infect. Immun. 68 (2000) 6912e6916. [4] W. Kreis, C. Hession, Isolation and purification of L-methionine-a-deamino-gmercaptomethane-lyase (L-methioninase) from Clostridium sporogenes, Cancer Res. 33 (1973) 1862e1865. [5] S.V. Revtovich, E.A. Morozova, N.V. Anufrieva, M.I. Kotlov, Y.F. Belyi, T.V. Demidkina, Identification of methionine g-lyase in genomes of some pathogenic bacteria, Dokl. Biochem. Biophys. 445 (2012) 187e193. [6] M. Tokoro, T. Asai, S. Kobayashi, T. Takeuchi, T. Nozaki, Identification and characterization of two isoenzymes of methionine g-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways, J. Biol. Chem. 278 (2003) 42717e42727. [7] B. Lockwood, G. Coombs, Purification and characterization of methionine glyase from Trichomonas vaginalis, Biochem. J. 279 (1991) 675e682. [8] F. Rebeille, S. Jabrin, R. Bligny, K. Loizeau, B. Gambonnet, V. Van Wilder, R. Douce, S. Ravanel, Methionine catabolism in Arabidopsis cells is initiated by a g-cleavage process and leads to S-methylcysteine and isoleucine syntheses, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15687e15692.
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