共查询到20条相似文献,搜索用时 15 毫秒
1.
Roi Feingersch Alon Philosof Tom Mejuch Fabian Glaser Onit Alalouf Yuval Shoham Oded Béjà 《The ISME journal》2012,6(4):827-834
Phosphonates (Pn) are diverse organic phosphorus (P) compounds containing C–P bonds and comprise up to 25% of the high-molecular weight dissolved organic P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary production in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a number of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn- as well as inorganic phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the number of Pn transporters with different specificities towards phosphite and different Pns. 相似文献
2.
Protein kinase A anchoring proteins (AKAPs), defined by their capacity to target the cAMP-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. Despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. Here, using bioinformatics and X-ray crystallography, we define a central domain of AKAP18δ (AKAP18CD) as a member of the 2H phosphoesterase family. The domain features two conserved His-x-Thr motifs positioned at the base of a groove located between two lobes related by pseudo 2-fold symmetry. Nucleotide co-crystallisation screening revealed that this groove binds specifically to adenosine 5'-monophosphate (5'AMP) and cytosine 5'-monophosphate (5'CMP), with the affinity constant for AMP in the physiological concentration range. This is the first example of an AKAP capable of binding a small molecule. Our data generate two functional hypotheses for the AKAP18 central domain. It may act as a phosphoesterase, although we did not identify a substrate, or as an AMP sensor with the potential to couple intracellular AMP levels to PKA signalling events. 相似文献
3.
ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of d-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases. 相似文献
4.
Ilari A Alaleona F Petrarca P Battistoni A Chiancone E 《Journal of molecular biology》2011,409(4):630-641
ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the cluster 9 group of ATP-binding cassette-type periplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, the ZnuABC system is essential for zinc uptake and homeostasis and is an important determinant of bacterial resistance to the host defense mechanisms. The cluster 9 members share a two (α/β)4 domain architecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by an insert of variable length, rich in histidine and negatively charged residues. This distinctive His-rich loop is proposed to play a role in the management of zinc also due to its location at the entrance of the metal binding site located at the domain interface. The known Synechocystis 6803 and Escherichia coli ZnuA structures show the same metal coordination involving three conserved histidines and a glutamic acid or a water molecule as fourth ligand. The structures of Salmonella enterica ZnuA, with a partially or fully occupied zinc binding site, and of a deletion mutant missing a large part of the His-rich loop revealed unexpected differences in the metal-coordinating ligands, as histidine 140 from the mobile (at the C-terminal) part of the loop substitutes the conserved histidine 60. This unforeseen coordination is rendered possible by the “open conformation” of the two domains. The possible structural determinants of these peculiarities and their functional relevance are discussed. 相似文献
5.
Imen Ferjani Abdellatif Fattoum Mohamed Manai Yves Benyamin Claude Roustan Sutherland K. Maciver 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(9):1760-1767
Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1. 相似文献
6.
Satoshi Akanuma 《Journal of molecular biology》2008,382(2):458-466
The (β/α)8-barrel is one of the most common folds functioning as enzymes. The emergence of two (β/α)8-barrel enzymes involved in histidine biosynthesis, each of which has a twofold symmetric structure, has been proposed to be a consequence of tandem duplication and fusion of a (β/α)4-half-barrel. However, little evidence has been found for the existence of an ancestral half-barrel in the evolution of other (β/α)8-barrel proteins. In order to detect remnants of an ancestral half-barrel in the (β/α)8-barrel structure of Escherichia coli N-(5′-phosphoribosyl)anthranilate isomerase, we engineered three potential half-barrel units, (β/α)1-4, (β/α)3-6, and (β/α)5-8. Among these three arrangements, only (β/α)3-6 is stable; it exists in equilibrium between monomeric and dimeric forms. Thus, the central segment of N-(5′-phosphoribosyl)anthranilate isomerase from E. coli can serve as a half-barrel precursor. A tandem duplication of (β/α)3-6 yielded predominantly monomeric structures that were quite stable. This result exemplified that the structural characteristics of noncovalently assembled half-barrels could be improved by duplication and fusion. Moreover, our results may provide information regarding the local structural units that encompass interactions important for the early folding events of this ubiquitous protein conformation. 相似文献
7.
The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (KD) of ∼ 0.29 and ∼ 29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle. 相似文献
8.
James A. Brannigan Zhiyong Yu Michael R. Hodgkinson Helen P. Price Robin J. Leatherbarrow Deborah F. Smith 《Journal of molecular biology》2010,396(4):985-999
N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovaniNMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 Å resolution. The structure has as its defining feature a 14-stranded twisted β-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis. 相似文献
9.
Daniel J. Koslover Furqan M. Fazal Rachel A. MooneyRobert Landick Steven M. Block 《Journal of molecular biology》2012,423(5):664-676
Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2 nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2 nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′ → 3′ toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins. 相似文献
10.
The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2′-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2′-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry. 相似文献
11.
Alasdair K. Mackenzie Karin Valegård Aman Iqbal Nadia J. Kershaw Christopher J. Schofield Inger Andersson 《Journal of molecular biology》2010,396(2):332-344
Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis. 相似文献
12.
The recently determined crystal structure of NhaA, the Na +/H + antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA. 相似文献
13.
The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca2+ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca2+ uptake and E–C coupling. Ca2+ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca2+ uptake as low as 5 °C. As a consequence, the Q10 values in coho salmon were low over a range of different temperature intervals. Maximal Ca2+ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca2+ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca2+ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function. 相似文献
14.
Frédéric Kerff Frédéric Mercier Raphaël Herman Astrid Zervosen Jean-Marie Frère Paulette Charlier 《Journal of molecular biology》2010,397(1):249-34403
AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-γ-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-γ-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases. 相似文献
15.
Ardeschir Vahedi-Faridi Anke Licht Haydar Bulut Sandro Keller Wolfram Saenger Erwin Schneider 《Journal of molecular biology》2010,397(3):709-105
GacH is the solute binding protein (receptor) of the putative oligosaccharide ATP-binding cassette transporter GacFG, encoded in the acarbose biosynthetic gene cluster (gac) from Streptomyces glaucescens GLA.O. In the context of the proposed function of acarbose (acarviosyl-1,4-maltose) as a ‘carbophor,’ the transporter, in complex with a yet to be identified ATPase subunit, is supposed to mediate the uptake of longer acarbose homologs and acarbose for recycling purposes. Binding assays using isothermal titration calorimetry identified GacH as a maltose/maltodextrin-binding protein with a low affinity for acarbose but with considerable binding activity for its homolog, component 5C (acarviosyl-1,4-maltose-1,4-glucose-1,1-glucose). In contrast, the maltose-binding protein of Salmonella typhimurium (MalE) displays high-affinity acarbose binding. We determined the crystal structures of GacH in complex with acarbose, component 5C, and maltotetraose, as well as in unliganded form. As found for other solute receptors, the polypeptide chain of GacH is folded into two distinct domains (lobes) connected by a hinge, with the interface between the lobes forming the substrate-binding pocket. GacH does not specifically bind the acarviosyl group, but displays specificity for binding of the maltose moiety in the inner part of its binding pocket. The crystal structure of acarbose-loaded MalE showed that two glucose units of acarbose are bound at the same region and position as maltose. A comparative analysis revealed that in GacH, acarbose is buried deeper into the binding pocket than in MalE by exactly one glucose ring shift, resulting in a total of 18 hydrogen-bond interactions versus 21 hydrogen-bond interactions for MalEacarbose. Since the substrate specificity of ATP-binding cassette import systems is determined by the cognate binding protein, our results provide the first biochemical and structural evidence for the proposed role of GacHFG in acarbose metabolism. 相似文献
16.
Carsten Kintscher 《Journal of molecular biology》2009,387(2):270-5145
Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg2+ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex. 相似文献
17.
Siu KK Lee JE Sufrin JR Moffatt BA McMillan M Cornell KA Isom C Howell PL 《Journal of molecular biology》2008,378(1):112-128
5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pKa of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN. 相似文献
18.
Satoru Shimizu Yoshiteru Sato Yu-ichiro Miyashita Kaoru Suzuki Masaru Tsunoda Anne-Catherine Dock-Bregeon Akio Takénaka 《Journal of molecular biology》2009,394(2):286-296
In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNAThr from different tRNAs in order to generate Thr-tRNAThr. In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNAThr, one for catalysis and the other for trans-editing of misacylated Ser-tRNAThr. In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 Å resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus. 相似文献
19.
Enzymes from the de novo purine biosynthetic pathway have been exploited for the development of anti-cancer drugs, and represent novel targets for anti-bacterial drug development. In Mycobacterium tuberculosis, the cause of tuberculosis, this pathway has been identified as essential for growth and survival. The structure of M. tuberculosis PurN (MtPurN) has been determined in complex with magnesium and iodide at 1.30 Å resolution, and with cofactor analogue, 5-methyltetrahydrofolate (5MTHF) at 2.2 Å resolution. The structure shows a Rossmann-type fold that is very similar to the known structures of the human and E. coli PurN proteins. In contrast, MtPurN forms a dimer that is quite different from that formed by the Escherichia coli PurN, and which suggests a mechanism whereby communication could take place between the two active sites. Differences are seen in two active site loops and in the binding mode of the 5MTHF cofactor analogue between the two MtPurN molecules of the dimer. A binding site for halide ions is found in the dimer interface, and bound magnesium and iodide ions in the active site suggest sites that might be exploited in potential drug discovery strategies. 相似文献
20.
Tomomi Sumida Ryohei Ishii Tatsuo Yanagisawa Makoto Ito 《Journal of molecular biology》2009,392(1):87-43012
We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids. 相似文献