共查询到20条相似文献,搜索用时 31 毫秒
1.
Spreitzer RJ 《Archives of biochemistry and biophysics》2003,414(2):141-149
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes. 相似文献
2.
Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action, observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity. 相似文献
3.
Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT)
Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (~160 kDa), responsible for methylation of DNA, and the restriction endonuclease (~400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT), based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7)TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NT)in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes. 相似文献
4.
5.
Michael A. Funk E. Neil G. Marsh Catherine L. Drennan 《The Journal of biological chemistry》2015,290(37):22398-22408
Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C—C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C—C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSβ, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these “X-succinate synthases” and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes. 相似文献
6.
Alejandra Herrera-Morande Victor Castro-Fernández Felipe Merino Cesar A. Ramírez-Sarmiento Francisco J. Fernández M. Cristina Vega Victoria Guixé 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(12):2869-2878
During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements. In ribokinase superfamily, two different topologies can be identified, which are related to each other as a non-cyclic permutation occurred during the evolution. Interestingly, this change in topology is correlated with the nucleotide specificity of its members. Thereby, the connectivity of the secondary elements allows us to distinguish an ATP-dependent and an ADP-dependent topology.Here we address the impact of introducing the topology of a homologous ATP-dependent kinase in an ADP-dependent kinase (Thermococcus litoralis glucokinase) in the structure, nucleotide specificity, and substrate binding order of the engineered enzyme. Structural evidence demonstrates that rewiring the topology of TlGK leads to an active and soluble enzyme without modifications on its three-dimensional architecture. The permuted enzyme (PerGK) retains the nucleotide preference of the parent TlGK enzyme but shows a change in the substrate binding order. Our results illustrate how the rearrangement of the protein folding topology during the evolution of the ribokinase superfamily enzymes may have dictated the substrate-binding order in homologous enzymes of this superfamily. 相似文献
7.
DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix-hairpin-helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases. 相似文献
8.
Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase. 相似文献
9.
Rein Kilkson 《Journal of biological physics》1989,17(2):103-107
Biomolecular structures are interacting in terms of their force fields. These force fields define the specificity surfaces of the molecules. Specificity surfaces are represented by specificity vectors in a multidimensional specificity space. A quantitative analytical expression is developed for biochemical reactions and the evolution of metabolic pathways in the specificity space. This leads to detailed identification of various biomolecular processes with individual terms in the equation. This theoretical analysis permits defining detailed function and resolution requirement of enzymes, as well as, how these fit into the overall metabolic pattern of the cell. This paper is Part II of a general theory of the physical basis of the biological state of matter. 相似文献
10.
C. R. Goward D. J. Nicholls 《Protein science : a publication of the Protein Society》1994,3(10):1883-1888
Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications. 相似文献
11.
Knecht W Sandrini MP Johansson K Eklund H Munch-Petersen B Piskur J 《The EMBO journal》2002,21(7):1873-1880
In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes. 相似文献
12.
Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity. 相似文献
13.
Summary Aminoacyl-tRNA synthetases are important components of the genetic apparatus. In spite of common catalytic properties, synthetases with different amino acid specificities are widely diverse in their primary structures, subunit sizes, and subunit composition. However, synthetases with given amino acid specificities are well conserved throughout evolution. We have been studying the human glutaminyl-tRNA synthetase possessing a sequence of about 400 amino acid residues (the core region) that is very similar to sequences in the corresponding enzymes from bacteria and yeast. The conserved sequence appears to be essential for the basic function of the enzyme, the charging of tRNA with glutamine. As a first step to a better understanding of the evolution of this enzyme, we determined the coding region for the conserved part of the human glutaminyl-tRNA synthetase. The coding region is composed of eight exons. It appears that individual exons encode defined secondary structural elements as parts of functionally important domains of the enzyme. Evolution of the gene by assembly of individual exons seems to be a viable hypothesis; alternative pathways are discussed.
Offprint requests to: R. Knippers 相似文献
14.
Emily K. LuebberingJacob Mick Ranjan K. SinghJohn J. Tanner Ritcha Mehra-ChaudharyLesa J. Beamer 《Journal of molecular biology》2012,423(5):831-846
The α‐d‐phosphohexomutase superfamily comprises enzymes involved in carbohydrate metabolism that are found in all kingdoms of life. Recent biophysical studies have shown for the first time that several of these enzymes exist as dimers in solution, prompting an examination of the oligomeric state of all proteins of known structure in the superfamily (11 different proteins; 31 crystal structures) via computational and experimental analyses. We find that these proteins range in quaternary structure from monomers to tetramers, with 6 of the 11 known structures being likely oligomers. The oligomeric state of these proteins not only is associated in some cases with enzyme subgroup (i.e., substrate specificity) but also appears to depend on domain of life, with the two archaeal proteins existing as higher‐order oligomers. Within the oligomers, three distinct interfaces are observed, one of which is found in both archaeal and bacterial proteins. Normal mode analysis shows that the topological arrangement of the oligomers permits domain 4 of each protomer to move independently as required for catalysis. Our analysis suggests that the advantages associated with protein flexibility in this enzyme family are of sufficient importance to be maintained during the evolution of multiple independent oligomers. This study is one of the first showing that global motions may be conserved not only within protein families but also across members of a superfamily with varying oligomeric structures. 相似文献
15.
The increasing sequence information on oxygen reductases of the haem-copper superfamily, together with the available three-dimensional structures, allows a clear identification of their common, functionally important features. Taking into consideration both the overall amino acid sequences of the core subunits and key residues involved in proton transfer, a novel hypothesis for the molecular evolution of these enzymes is proposed. Three main families of oxygen reductases are identified on the basis of common features of the core subunits, constituting three lines of evolution: (i) type A (mitochondrial-like oxidases), (ii) type B (ba3-like oxidases) and (iii) type C (cbb3-type oxidases). The first group can be further divided into two subfamilies, according to the helix VI residues at the hydrophobic end of one of the proton pathways (the so-called D-channel): (i) type A1, comprising the enzymes with a glutamate residue in the motif -XGHPEV-, and (ii) type A2, enzymes having instead a tyrosine and a serine in the alternative motif -YSHPXV-. This second subfamily of oxidases is shown to be ancestor to the one containing the glutamate residue, which in the Bacteria domain is only present in oxidases from Gram-positive or purple bacteria. It is further proposed that the Archaea domain acquired terminal oxidases by gene transfer from the Gram-positive bacteria, implying that these enzymes were not present in the last common ancestor before the divergence between Archaea and Bacteria. In fact, most oxidases from archaea have a higher amino acid sequence identity and similarity with those from bacteria, mainly from the Gram-positive group, than with oxidases from other archaea. Finally, a possible relation between the dihaemic subunit (FixP) of the cbb3 oxidases and subunit II of caa3 oxidases is discussed. As the families of haem-copper oxidases can also be identified by their subunit II, a parallel evolution of subunits I and II is suggested. 相似文献
16.
Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine 总被引:1,自引:0,他引:1
A new adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), has been synthesized. The effectiveness of PLP-AMP as an affinity probe has been tested using a number of nucleotide-binding enzymes. In comparison to reaction with pyridoxal 5'-phosphate, PLP-AMP binds more tightly and exhibits greater specificity of labeling for most enzymes tested. PLP-AMP is a very potent inhibitor of yeast alcohol dehydrogenase and rabbit muscle pyruvate kinase, with complete inhibition obtained upon incorporation of 1 mol of reagent/mol of catalytic subunit. The reagent is also a potent inhibitor of yeast hexokinase and phosphoglycerate kinase. When modified in the absence of substrates, these enzymes require 2 mol of reagent/mol of active site for complete inhibition. However, when modified in the presence of sugar substrates, this stoichiometry decreases to 1.1 for the hexokinase-glucose complex and 1.4 for the phosphoglycerate kinase . 3-phosphoglycerate complex. The most potent inhibition by PLP-AMP was observed with rabbit muscle adenylate kinase. Half-maximal inhibition was obtained at a concentration of approximately 1 microM. In contrast to these examples, PLP-AMP, as well as pyridoxal 5'-phosphate, fails to act as a potent or specific inhibitor of beef heart mitochondrial F1-ATP-ase. The high specificity of labeling and the ability of nucleotide substrates to decrease the rate of inactivation of the kinases and dehydrogenase are consistent with the modification of active site residues. The complete reversibility of both modification and inactivation in the absence of reduction by NaBH4 and the absorption spectra of modified enzymes prior to and following reduction indicate reaction with lysyl residues. We conclude that PLP-AMP holds considerable promise as an affinity label for exploring the structure and mechanism of nucleotide-binding enzymes. 相似文献
17.
Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures
or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human,
Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one
or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements
is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in
animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative
oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the
first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second
oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions
at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules
M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide
segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins
have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact.
Received: 12 January 2001 / Accepted: 3 April 2001 相似文献
18.
Kaushal Parikh Sander H. Diks Jurriaan H. B. Tuynman Auke Verhaar Mark L?wenberg Daan W. Hommes Jos Joore Akhilesh Pandey Maikel P. Peppelenbosch 《PloS one》2009,4(7)
Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events. 相似文献
19.
Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF BP) or the gamma subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF-BP or the gamma subunit of 7SNGF. The hydrolysis id pH dependent with maximum proteolysis at pH 6.0-7.0. the newly formed C-terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2-4 microgram/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin-like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event. 相似文献