Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties.     

Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver

Liver-specific and non-liver-specific methionine adenosyltransferase (MAT) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (SAM). We previously showed that MAT2A expression was associated with more rapid cell growth. Changes in MAT expression have not been examined in animal models of alcoholic liver injury, which is the focus of the current study. After rats were fed intragastrically with ethanol and high fat for 9 wk, the mRNA level of both MAT forms doubled but only the protein level of MAT2A increased. Although liver-specific MAT activity did not change, it was 32% lower after one and 68% lower after eight weekly enteral doses of lipopolysaccharide. Hepatic levels of methionine, SAM, and DNA methylation fell by approximately 40%. c-myc was hypomethylated, and its mRNA level increased. Genome-wide DNA strand break increased. Thus in the prefibrotic stage of alcoholic liver injury, there is already a switch in MAT expression, global DNA hypomethylation, increased c-myc expression, and genome-wide DNA strand break. These changes may be important in predisposing this liver disease to malignant degeneration.     

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase.

Rat liver methionine adenosyltransferase incorporated 8 mol of N-ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.     

Refolding and characterization of methionine adenosyltransferase from Euglena gracilis

Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 μmol/min/mg and K(m) values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.     

Mutational analysis of methionine adenosyltransferase from Leishmania donovani.

The methionine adenosyltransferase (MAT; EC 2.5.1.6) mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process, consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities--AdoMet synthesis and tripolyphosphate hydrolysis--can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. This report describes the mutational analysis of the amino acids involved in both the ATP and L-methionine binding sites of Leishmania donovani MAT (GenBank accession number AF179714) the aetiological agent of visceral leishmaniasis. Site-directed mutagenesis was used to substitute neutral residues for the basic amino acid (Lys168, Lys256, Lys276, Lys280 and His17), acidic residues (Asp19, Asp121, Asp166, Asp249, Asp277 and Asp288) and Phe241 involved in AdoMet synthesis and PPPi hydrolysis. With the exception of D116N, none of these mutants was able to synthesize AdoMet at a significant rate, although H17A, H17N, K256A, K280A, D19N, D121N, D166N, D249N and D282N showed measurable tripolyphosphatase activity. Finally, the C-terminus domain of L. donovani MAT was truncated at three points (F382Stop, D375Stop, F368Stop), deleting a 3(10) one-turn helix motif in all three cases. Whilst none of the truncated proteins conserved MAT activity, they were able to hydrolyse PPPi, albeit at a lower rate than the wild-type enzyme. A fourth protein with an internal deletion (E376DeltaF382) in the C-terminal domain conserved high tripolyphosphatase activity, which was not, however, induced by 50 microM AdoMet.     

The three-dimensional structure of diaminopimelate decarboxylase from Mycobacterium tuberculosis reveals a tetrameric enzyme organisation

The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 Å. The monoclinic crystals contain one tetramer exhibiting D₂-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer–tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.     

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.     

The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features.

Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented.     

The crystal structure of alpha-thrombin-hirunorm IV complex reveals a novel specificity site recognition mode

The X-ray crystal structure of the human alpha-thrombin-hirunorm IV complex has been determined at 2.5 A resolution, and refined to an R-factor of 0.173. The structure reveals an inhibitor binding mode distinctive of a true hirudin mimetic, which justifies the high inhibitory potency and the selectivity of hirunorm IV. This novel inhibitor, composed of 26 amino acids, interacts through the N-terminal end with the alpha-thrombin active site in a nonsubstrate mode, and binds specifically to the fibrinogen recognition exosite through the C-terminal end. The backbone of the N-terminal tripeptide Chg1"-Arg2"-2Na13" (Chg, cyclohexyl-glycine; 2Na1, beta-(2-naphthyl)-alanine) forms a parallel beta-strand to the thrombin main-chain segment Ser214-Gly216. The Chg1" side chain occupies the S2 site, Arg2" penetrates into the S1 specificity site, while the 2Na13" side chain occupies the aryl binding site. The Arg2" side chain enters the S1 specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2" side chain establishes the typical ion pair with the carboxylate group of Asp189.     

AFM imaging reveals the tetrameric structure of the TRPM8 channel

Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm³, close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93° and 172°, confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily.     

The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site

Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 Å resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.     

The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity

The I-SceI homing endonuclease enhances gene targeting by introducing double-strand breaks at specific chromosomal loci, thereby increasing the recombination frequency. Here, we report the crystal structure of the enzyme complexed to its DNA substrate and Ca(2+) determined at 2.25A resolution. The structure shows the prototypical beta-saddle of LAGLIDADG homing endonucleases that is contributed by two pseudo-symmetric domains. The high specificity of I-SceI is explained by the large number of protein-DNA contacts, many that are made by a long beta-hairpin loop that reaches into the major groove of the DNA. The DNA minor groove is compressed at the catalytic center, bringing the two scissile phosphodiester bonds into close proximity. The protein-Ca(2+)-DNA structure shows the protein bound to its DNA substrate in a pre-reactive state that is defined by the presence of two asymmetric active sites, one of which appears poised to first cleave the DNA bottom strand.     

The crystal structure of a (-) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site

The structure of the recombinant (-) gamma-lactamase from an Aureobacterium species has been solved at 1.73A resolution in the cubic space group F23 with unit cell parameters a=b=c=240.6A. The trimeric enzyme has an alpha/beta hydrolase fold and closely resembles the cofactor free haloperoxidases. The structure has been solved in complex with a covalently bound ligand originating from the host cell and also in the unligated form. The associated density in the former structure has been interpreted as the two-ring ligand (3aR,7aS)-3a,4,7,7a-tetrahydro-benzo [1,3] dioxol-2-one which forms a tetrahedral complex with OG of the catalytic Ser98. Soaks of these crystals with the industrial substrate gamma-lactam or its structural analogue, norcamphor, result in the displacement of the ligand from the enzyme active site, thereby allowing determination of the unligated structure. The presence of the ligand in the active site protects the enzyme from serine hydrolase inhibitors. Cyclic ethylene carbonate, the first ring of the ligand, was shown to be a substrate of the enzyme.     

The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site

The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.