Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

LinA is the first enzyme of the microbial degradation pathway of a chlorinated insecticide, hexachlorocyclohexane (HCH), and mediates the dehydrochlorination of α-, γ-, and δ-HCH. Its two variants, LinA type 1 and LinA type 2, which differ at 10 out of 156 amino acid residues, have been described. Their activities for the metabolism of different HCH isomers differ considerably but overall are high for γ-HCH, moderate for α-HCH, low for δ-HCH, and lacking for β-HCH. Here, we describe the characterization of a new variant of this enzyme, LinA type 3, whose gene was identified from the metagenome of an HCH-contaminated soil sample. Its deduced primary structure in the region spanning amino acid residues 1 to 147 of the protein exhibits 17 and 12 differences from LinA type 1 and LinA type 2, respectively. In addition, the residues GIHFAPS, present at the region spanning residues 148 to 154 in both LinA type 1 and LinA type 2, are deleted in LinA type 3.The activity of LinA type 3 for the metabolism of δ-HCH is several orders of magnitude higher than that of LinA type 1 or LinA type 2 and can be useful for improvement of the metabolism of δ-HCH.     

Degradation of β-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

β-Hexachlorocyclohexane (β-HCH) is the most recalcitrant among the α-, β-, γ-, and δ-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of γ-HCH in Sphingomonas paucimobilis UT26, metabolizes β-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

Enantioselective Dehydrochlorination of δ-Hexachlorocyclohexane and δ-Pentachlorocyclohexene by LinA1 and LinA2 from Sphingobium indicum B90A

δ-Hexachlorocyclohexane (δ-HCH), one of the prevalent isomers of technical HCH, was enantioselectively dehydrochlorinated by the dehydrochlorinases LinA1 and LinA2 from Sphingobium indicum B90A to the very same δ-pentachlorocyclohexene enantiomer. Racemic δ-pentachlorocyclohexene, however, was transformed with opposite enantioselectivities by the two enzymes. A transformation pathway based on an anti-1,2-elimination, followed by a syn-1,4-elimination and a subsequent syn-1,2-elimination is postulated.     

Crystal Structure and Putative Mechanism of 3-Methylitaconate-Δ-isomerase from Eubacterium barkeri

3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K_m but > 1000-fold lower k_cat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P2₁2₁2₁ unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.     

Crystal Structure of the Resuscitation-Promoting Factor ΔDUFRpfB from M. tuberculosis

Mycobacterium tuberculosis is able to establish a non-replicating state and survive in an intracellular habitat for years. Resuscitation of dormant M. tuberculosis bacteria is promoted by resuscitation-promoting factors (Rpfs), which are secreted from slowly replicating bacteria close to dormant bacteria. Here we report the crystal structure of a truncated form of RpfB (residues 194-362), the sole indispensable Rpf of the five Rpfs encoded in this bacterium genome. The structure, denoted as _ΔDUFRpfB, exhibits a comma-like shape formed by a lysozyme-like globular catalytic domain and an elongated G5 domain, which is widespread among cell surface binding proteins. The G5 domain, whose structure was previously uncharacterised, presents some peculiar features. The basic structural motif of this domain, which represents the tail of the comma-like structure, is a novel super-secondary-structure element, made of two β-sheets interconnected by a pseudo-triple helix. This intricate organisation leads to the exposure of several backbone hydrogen-bond donors/acceptors. Mutagenesis analyses and solution studies indicate that this protein construct as well as the full-length form are elongated monomeric proteins. Although _ΔDUFRpfB does not self-associate, the exposure of structural elements (backbone H-bond donors/acceptors and hydrophobic side chains) that are usually buried in globular proteins is typically associated with adhesive properties. This suggests that the RpfB G5 domain has a cell-wall adhesive function, which allows the catalytic domain to be properly oriented for the cleavage reaction. Interestingly, sequence comparisons indicate that these structural features are also shared by G5 domains involved in biofilm formation.     

The Crystal Structure of Protein MJ1225 from Methanocaldococcus jannaschii Shows Strong Conservation of Key Structural Features Seen in the Eukaryal γ-AMPK

In mammals, 5′-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (α) and two regulatory subunits (β and γ). The γ-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the γ-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine β-synthase domains so far. Site E is located in a small cavity existing between the α-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in γ-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.     

Anion activation of γ-glutamyltransferase from fruiting bodies of Lentinus edodes

Activation by different anions of γ-glutamyltransferase obtained in a. particulate form from fruiting bodies of Lentinus edodes has been studied using either L-γ-glutamyl-p-nitroanlide or lentinic acid as substrate. The mushroom transferase was activated by SCN^?, NO₃^?, Cl^?, Br^?, ClO₃^?, Bro₃^?, N₃^?, I^? and F^?, but not those alkali and earth cations previously believed to activate the animal transferase, nor by citrate, claimed to be effective for the kidney bean transferase. Among anions proved hardly to activate the transferase were ClO₄^?, NO₂?, HCO₃^?, H₂PO₄^?, SO₃^2? and SO₄^2?. A high concentration of these anions more or less impeded the halide activation. Kinetic studies revealed that halides function as activators of increasing V_max while keeping K_m constant. These observations appeared least compatible with the possibility that the anion activation might involve a non-specific effect of high solute concentration, viz. dissociation of the enzyme from the supporting structure in the particulates. The activating effect of halides described here probably extends also to the animal enzymes.     

Corrinoids from Methanosarcina barkeri: Structure of the α-ligand

5-Hydroxybenzimidazole is the only base detected in cobamide compounds from methanol-grown Methanosarcina barkeri. 5-Hydroxybenzimidazolylcobamide accounted for about 83 and 90% of the total corrinoids of whole cells and cell-free extracts, respectively. Probably, the rest of the corrinoids are base-less.     

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

The enzyme γ-glutamyltranspeptidase 1 (GGT1) is a conserved member of the N-terminal nucleophile hydrolase family that cleaves the γ-glutamyl bond of glutathione and other γ-glutamyl compounds. In animals, GGT1 is expressed on the surface of the cell and has critical roles in maintaining cysteine levels in the body and regulating intracellular redox status. Expression of GGT1 has been implicated as a potentiator of asthma, cardiovascular disease, and cancer. The rational design of effective inhibitors of human GGT1 (hGGT1) has been delayed by the lack of a reliable structural model. The available crystal structures of several bacterial GGTs have been of limited use due to differences in the catalytic behavior of bacterial and mammalian GGTs. We report the high resolution (1.67 Å) crystal structure of glutamate-bound hGGT1, the first of any eukaryotic GGT. Comparisons of the active site architecture of hGGT1 with those of its bacterial orthologs highlight key differences in the residues responsible for substrate binding, including a bimodal switch in the orientation of the catalytic nucleophile (Thr-381) that is unique to the human enzyme. Compared with several bacterial counterparts, the lid loop in the crystal structure of hGGT1 adopts an open conformation that allows greater access to the active site. The hGGT1 structure also revealed tightly bound chlorides near the catalytic residue that may contribute to catalytic activity. These are absent in the bacterial GGTs. These differences between bacterial and mammalian GGTs and the new structural data will accelerate the development of new therapies for GGT1-dependent diseases.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.     

Complete Genome Sequence of the Representative γ-Hexachlorocyclohexane-Degrading Bacterium Sphingobium japonicum UT26

Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.γ-Hexachlorocylohexane (γ-HCH) is a man-made insecticide that has caused serious environmental problems worldwide (9). Although only several decades have passed since the initial release of γ-HCH into the environment, many γ-HCH-degrading bacterial strains have been isolated (9), suggesting that γ-HCH-degrading ability was acquired by such strains within a short period (14). Sphingobium japonicum UT26 was isolated from upland γ-HCH-contaminated soil in Japan and utilizes γ-HCH as its sole source of carbon and energy (8, 16). In UT26, 15 lin genes are involved in γ-HCH utilization (2, 11, 12). Our clarification of the complete genome sequence of this strain is expected to provide insights into the mechanisms by which bacteria adapt to xenobiotics.One S. japonicum UT26 colony was designated UT26S (NBRC 101211), and its complete genome sequence was determined by a whole-genome shotgun sequencing strategy using the Sanger method (10, 15, 17). The sequences of ca. 92,400 reads were assembled by the Phrap and CONSED assembly tools (4, 5, 7), and the gaps between contigs were closed by sequencing PCR products which were amplified from genomic DNA using the appropriate primers. The prediction and annotation of open reading frames (ORFs) were performed using Glimmer3 (1), BLASTP, the In Silico Molecular Cloning software suite (In Silico Biology, Inc.), and the GenomeMatcher software (13). Nontranslating genes were predicted using the Rfam, tRNAscan-SE, and ARAGORN programs.The genome of S. japonicum UT26S consists of two circular chromosomes (Chr), Chr 1 (3,514,822 bp, 64.8% G+C, 3,529 ORFs) and Chr 2 (681,892 bp, 65.9% G+C, 589 ORFs), and three circular plasmids, pCHQ1 (190,974 bp, 63.0% G+C, 224 ORFs), pUT1 (31,776 bp, 63.7% G+C, 44 ORFs), and pUT2 (5,398 bp, 61.0% G+C, 8 ORFs). Chr 1 and Chr 2 have one and two copies, respectively, of rRNA operons. Fifty-one and 4 tRNA genes were located on Chr 1 and Chr 2, respectively. One hundred ninety-six out of 206 bacterial essential genes proposed by Gil et al. (6) were all located on Chr 1, indicating that this is clearly a “main” chromosome. The 15 lin genes for γ-HCH degradation are dispersed on Chr 1, Chr 2, and pCHQ1. Comparison of the UT26S genome with those of five other sphingomonad (TM1) strains revealed that the lin genes (linA, linB, linC, linRED, and linF) specific for the conversion of γ-HCH to β-ketoadipate are located in the DNA regions unique to the UT26S genome. On the other hand, linGHIJ for the β-ketoadipate pathway (12) and linKLMN for the ABC transporter system (3) are located in conserved genomic regions of these sphingomonads. Based on these results, we propose a model in which UT26S was established by recruiting the specific lin genes into an ancestral strain having core functions of sphingomonads.     

Crystal Structure of the TNF-α-Inducing Protein (Tipα) from Helicobacter pylori: Insights into Its DNA-Binding Activity

Helicobacter pylori infection is one of the highest risk factors for gastroduodenal diseases including gastric cancer. Tumor necrosis factor-alpha (TNF-α) is one of the essential cytokines for tumor promotion, and thus, an H. pylori protein that induces TNF-α is believed to play a significant role in gastric cancer development in humans. The HP0596 gene product of H. pylori strain 26695 was identified as the TNF-α-inducing protein (Tipα). Tipα is secreted from H. pylori as dimers and enters the gastric cells. It was shown to have a DNA-binding activity. Here, we have determined the crystal structure of Tipα from H. pylori. Its monomer consists of two structural domains (“mixed domain” and “helical domain”). Tipα exists as a dimer in the crystal, and the dimeric structure represents a novel scaffold for DNA binding. A positively charged surface patch formed across the two monomers of the Tipα dimer by the loop between helices α1 and α2 may be important in DNA binding.     

Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ?-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ?-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT’s hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates.

Abbreviations: ?-glutamyl peptide, theanine, X-ray crystallography.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Structure and function of γ-secretase

The γ-secretase complex is a prime target for pharmacological intervention in Alzheimer’s disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as γ-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about γ-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of γ-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.     

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.     

Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.     

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

Microtubules are composed of polymerized α/β-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures β-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with β-tubulin in plant. Furthermore, mutagenesis studies indicated that the α-helical regions of KIS participate in β-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to β-tubulin in plants.

Structured summary

MINT-7968902, MINT-7968915, MINT-7968951, MINT-7968966: KIS (uniprotkb:O04350) physically interacts (MI:0915) with Tub9 (uniprotkb:P29517) by anti tag coimmunoprecipitation (MI:0007)MINT-7968928: KIS (uniprotkb:O04350) and Tub9 (uniprotkb:P29517) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)