Cryo-EM structure of I domain-containing integrin αEß7.

The integrin family is a transmembrane receptor that plays critical roles in the cell-cell and cell-extracellular matrix adhesion, signal transduction such as cell cycle regulation, organization of the intracellular cytoskeleton, and immune responses. Consequently, dysfunction of integrins is associated with a wide range of human diseases, including cancer and immune diseases, which makes integrins therapeutic targets for drug discovery. Here we report the cryo-EM structure of the human α-I domain-containing full-length integrin αEß7, which is expressed in the leukocytes of the immune system and a drug target for inflammatory bowel disease (IBD). The structure reveals the half-bent conformation, an intermediate between the close and the open conformation, while the α-I domain responsible for the ligand binding covers the headpiece domain by a unique spatial arrangement. Our results provide the structural information for the drug design targeting IBD.

Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria.

Recent studies on the respiratory chain of Ascaris suum showed that the mitochondrial NADH-fumarate reductase system composed of complex I, rhodoquinone and complex II plays an important role in the anaerobic energy metabolism of adult A. suum. The system is the major pathway of energy metabolism for adaptation to a hypoxic environment not only in parasitic organisms, but also in some types of human cancer cells. Thus, enzymes of the pathway are potential targets for chemotherapy. We found that flutolanil is an excellent inhibitor for A. suum complex II (IC50 = 0.058 µM) but less effectively inhibits homologous porcine complex II (IC50 = 45.9 µM). In order to account for the specificity of flutolanil to A. suum complex II from the standpoint of structural biology, we determined the crystal structures of A. suum and porcine complex IIs binding flutolanil and its derivative compounds. The structures clearly demonstrated key interactions responsible for its high specificity to A. suum complex II and enabled us to find analogue compounds, which surpass flutolanil in both potency and specificity to A. suum complex II. Structures of complex IIs binding these compounds will be helpful to accelerate structure-based drug design targeted for complex IIs.

Mechanically Tunable Hydrogels with Self-Healing and Shape Memory Capabilities from Thermo-Responsive Amino Acid-Derived Vinyl Polymers.

In this study, we report the fabrication and characterization of self-healing and shape-memorable hydrogels, the mechanical properties of which can be tuned via post-polymerization crosslinking. These hydrogels were constructed from a thermo-responsive poly(N-acryloyl glycinamide) (NAGAm) copolymer containing N-acryloyl serine methyl ester (NASMe) units (5 mol%) that were readily synthesized via conventional radical copolymerization. This transparent and free-standing hydrogel is produced via multiple hydrogen bonds between PNAGAm chains by simply dissolving the polymer in water at a high temperature (~90 °C) and then cooling it. This hydrogel exhibited moldability and self-healing properties. The post-polymerization crosslinking of the amino acid-derived vinyl copolymer network with glutaraldehyde, which acts as a crosslinker between the hydroxy groups of the NASMe units, tuned mechanical properties such as viscoelasticity and tensile strength. The optimal crosslinker concentration efficiently improved the viscoelasticity. Moreover, these hydrogels exhibited shape fixation (~60%)/memory (~100%) behavior owing to the reversible thermo-responsiveness (upper critical solution temperature-type) of the PNAGAm units. Our multifunctional hydrogel, with moldable, self-healing, mechanical tunability via post-polymerization crosslinking, and shape-memorable properties, has considerable potential for applications in engineering and biomedical materials.

Characterization of Information-Transmitting Materials Produced in Ionic Liquid-based Neuromorphic Electrochemical Devices for Physical Reservoir Computing.

Device implementation of reservoir computing, which is expected to enable high-performance data processing in simple neural networks at a low computational cost, is an important technology to accelerate the use of artificial intelligence in the real-world edge computing domain. Here, we propose an ionic liquid-based physical reservoir device (IL-PRD), in which copper cations dissolved in an IL induce diverse electrochemical current responses. The origin of the electrochemical current from the IL-PRD was investigated spectroscopically in detail. After operating the device under various operating conditions, X-ray photoelectron spectroscopy of the IL-PRD revealed that electrochemical reactions involving Cu, Cu2O, Cu(OH)2, CuSx, and H2O occur at the Pt electrode/IL interface. These products are considered information transmission materials in IL-PRD similar to neurotransmitters in biological neurons. By introducing the Faradaic current components due to the electrochemical reactions of these materials into the output signal of IL-PRD, we succeeded in improving the time-series data processing performance of the nonlinear autoregressive moving average task. In addition, the information processing efficiency in machine learning to classify electrocardiogram signal waveforms was successfully improved by using the output current from IL-PRD. Optimizing the electrochemical reaction products of IL-PRD is expected to advance data processing technology in society.

Transcriptional and functional analysis of trifluoromethionine resistance in Entamoeba histolytica.

OBJECTIVES: Drug resistance in parasitic protozoa is an obstacle to successful chemotherapy. Understanding how pathogens respond to drugs is crucial in preventing resistance. Previously, we have shown that in Entamoeba histolytica, methionine γ-lyase (EhMGL) downregulation results in trifluoromethionine resistance. The transcriptional response, however, of this parasite to the drug is not known. In this study, we used microarray analysis to determine whether additional genes are involved. METHODS: The expression profiles of 9230 genes in wild-type and trifluoromethionine-resistant strains were compared. Episomal overexpression of EhBspA1 was performed to verify its role in trifluoromethionine resistance. The transcriptomes of a trifluoromethionine-resistant strain cultured with or without trifluoromethionine, an EhMGL gene-silenced strain, a strain with reduced susceptibility to metronidazole and a wild-type strain under cysteine-deprived conditions were compared to determine the specificity of the changes observed in the trifluoromethionine-resistant strain. RESULTS: The expression of 35 genes differed at least 3-fold between trifluoromethionine-resistant and wild-type strains. Some of the genes play roles in metabolism, the stress response and gene regulation. EhMGL and EhBspA1 were found to be highly downregulated and upregulated, respectively. Overexpression of EhBspA1 conferred partial resistance to trifluoromethionine. Comparative transcriptome analysis showed that genes modulated in trifluoromethionine-resistant strains were specific. CONCLUSIONS: E. histolytica has few known resistance mechanisms against drugs. In this study, we showed that aside from EhMGL downregulation, induction of EhBspA1 plays a role in trifluoromethionine resistance. We also showed a unique set of induced genes that could represent the signature profile of trifluoromethionine resistance in E. histolytica.

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel ß-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

Dynamic Nonlinear Behavior of Ionic Liquid-Based Reservoir Computing Devices.

Herein, a physical reservoir device that uses faradaic currents generated by redox reactions of metal ions in ionic liquids was developed. Synthetic time-series data consisting of randomly arranged binary number sequences ("1" and "0") were applied as isosceles-triangular voltage pulses with positive and negative voltage heights, respectively, and the effects of the faradaic current on short-term memory and parity-check task accuracies were verified. The current signal for the first half of the triangular voltage-pulse period, which contained a much higher faradaic current component compared to that of the second half of the triangular voltage-pulse period, enabled higher short-term memory task accuracy. Furthermore, when parity-check tasks were performed using a faradaic current generated by asymmetric triangular voltage-pulse levels of 1 and 0, the parity-check task accuracy was approximately eight times higher than that of the symmetric triangular voltage pulse in terms of the correlation coefficient between the output signal and target data. These results demonstrate the advantage of the faradaic current on both the short-term memory characteristics and nonlinear conversion capabilities and are expected to provide guidance for designing and controlling various physical reservoir devices that utilize electrochemical reactions.

Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cystine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica.

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Metabolome analysis revealed increase in S-methylcysteine and phosphatidylisopropanolamine synthesis upon L-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica.

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

Global analysis of gene expression in response to L-Cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. E. histolytica completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in E. histolytica, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism. RESULTS: In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion. CONCLUSIONS: To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol.

Mechanism of trifluoromethionine resistance in Entamoeba histolytica.

OBJECTIVES: To determine the mechanism of trifluoromethionine resistance in Entamoeba histolytica and evaluate the impact of acquired drug resistance on virulence. METHODS: Trifluoromethionine-resistant amoebae were selected in vitro and examined for cross-resistance to antiamoebic drugs, stability of resistance, methionine γ-lyase (MGL) activity, cell adhesion and virulence. Targeted gene silencing was performed to confirm the role of EhMGL. RESULTS: Trophozoites with a resistance index of 154 were obtained. The cells were susceptible to chloroquine, metronidazole, paromomycin and tinidazole, but remained resistant to trifluoromethionine in the absence of drug pressure. A complete lack of EhMGL activity accompanied by increased adhesion and decreased cytolysis were also observed. Silencing of the EhMGL genes resulted in trifluoromethionine resistance. CONCLUSIONS: This study provides the first demonstration of trifluoromethionine resistance in a parasitic protozoon. Repression of gene expression of drug targets represents a novel mechanism of resistance in E. histolytica. The information obtained from this work should help further development of trifluoromethionine derivatives that have lower chances of inducing resistance.

Structural basis for substrate specificity of l-methionine decarboxylase.

l -Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B6 -dependent enzyme and catalyzes the non-oxidative decarboxylation of l -methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

Methionine gamma-lyase: the unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers.

Sulfur-containing amino acids (SAAs) are essential components in many biological processes and ubiquitously distributed to all organisms. Both biosynthetic and catabolic pathways of SAAs are heterogeneous among organisms and between developmental stages, and regulated by the environmental changes. Limited lineage of organisms ranging from archaea to plants, but not human, possess a unique enzyme methionine gamma-lyase (MGL, EC 4.4.1.11) to directly degrade SAA to alpha-keto acids, ammonia, and volatile thiols. The reaction mechanisms and the physiological roles of this enzyme are partially demonstrated by the enzymological analyzes, structure determination, isotopic labeling of the intermediate metabolites, and functional analyzes of deficient mutants. MGL has been exploited as a drug target for the infectious diseases caused by parasitic protozoa and anaerobic periodontal bacteria. In addition, MGL has been utilized to develop therapeutic interventions of various cancers, by introducing recombinant proteins to deplete methionine essential for the growth of cancer cells. In this review, we discuss the current understanding of enzymological properties, putative physiological roles, and therapeutic applications of MGL.

Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis.

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.

Crystallization and preliminary X-ray analysis of L-methionine gamma-lyase 1 from Entamoeba histolytica.

L-Methionine gamma-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2(1), with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 A, beta = 101.82 degrees . The crystals diffract to beyond 2.0 A resolution. The presence of a tetramer in the asymmetric unit (4 x 42.4 kDa) gives a Matthews coefficient of 2.8 A(3) Da(-1) and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite.

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.

Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica.

The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

X-Ray snapshots of a pyridoxal enzyme: a catalytic mechanism involving concerted [1,5]-hydrogen sigmatropy in methionine γ-lyase.

Pyridoxal 5'-phosphate (PLP)-enzymes are essentially involved in amino acid and amine metabolism of a wide variety of organisms. Despite their extensive biochemical studies, there are little evidence and structural data to comprehensively elaborate the catalytic mechanism. We obtained X-ray snapshots of l-methionine γ-lyase from Entamoeba histolytica (EhMGL), a PLP-enzyme catalyzing the γ-elimination reaction of methionine. Here, we suggest a catalytic mechanism of EhMGL by using the X-ray snapshots covering all stages of this multistep catalysis reaction. Initial formation of a Michaelis complex is followed by the migration of double bond from the C4'=Nα-Cα moiety in an intermediate PLP-methionine imine to C4'-Nα=Cα in pyridoxamine 5'-phosphate (PMP)-α,ß-dehydromethionine imine without intervention of a putative quinonoid intermediate. The enzyme can facilitate the subsequent γ-elimination of methanethiol by the possible general acid-base catalysis of Tyr108 for the E1cB mechanism, enabling to form the ene-imine C4'-Nα=Cα-Cß=Cγ structure with the s-cis conformation, which is prerequisite for the non-enzymatic symmetry-allowed suprafacial [1,5]-hydrogen shift to complete the catalytic cycle by releasing α-ketobutyrate. The mechanism based on the X-ray snapshots is consistent with the reactivity of MGL toward methionine analogues. The generality of such a mechanism involving non-enzymatic concerted reaction in other PLP enzymes is discussed.

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis.

Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, ß-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.