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1.
The (betaalpha)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (betaalpha) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consists of the four N-terminal (betaalpha) units and HisF-C of the four C-terminal (betaalpha) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (betaalpha)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.  相似文献   

2.
Natural molecular evolution supplies us with manifold examples of protein engineering. The imitation of these natural processes in the design of new enzymes has led to surprising and insightful results. Well-suited for design by evolutionary methods are enzymes with the common and versatile (betaalpha)(8)-barrel fold. Studies of enzyme stability, folding and design as well as the evolution of (betaalpha)(8)-barrel enzymes are discussed.  相似文献   

3.
The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the existing evolutionary division of the entire family of (alpha/beta)8-barrel proteins.  相似文献   

4.
A common evolutionary origin of two elementary enzyme folds   总被引:1,自引:0,他引:1  
The (beta alpha)(8)-barrel is the most frequent and most versatile fold among enzymes [H?cker et al., Curr. Opin. Biotechnol. 12 (2001) 376-381; Wierenga, FEBS Lett. 492 (2001) 193-198]. Structural and functional evidence suggests that (beta alpha)(8)-barrels evolved from an ancestral half-barrel, which consisted of four (beta alpha) units stabilized by dimerization [Lang et al., Science 289 (2000) 1546-550; H?cker et al., Nat. Struct. Biol. 8 (2001) 32-36; Gerlt and Babbitt, Nat. Struct. Biol. 8 (2001) 5-7]. Here, by performing a comprehensive database search, we detect a striking and unexpected structural and amino acid sequence similarity between (beta alpha)(4) half-barrels and members of the (beta alpha)(5) flavodoxin-like fold. These findings provoke the hypothesis that a large fraction of the modern-day enzymes evolved from a basic structural building block, which can be identified by a combination of sequence and structural analyses.  相似文献   

5.
Two sequence-related subfamilies of flavin-binding β/α-barrels have been identified (the type I and type II proteins) that differ in the nature of residue packing in the core of the barrel domain. Similar observed differences in the packing of internal amino acid side chains in β/α-barrels have previously been used to argue that these domains have evolved convergently toward a stable structural framework. Using structural alignments of flavin-binding barrel proteins, we demonstrate that simple genetic alterations may be responsible for switching the nature of side-chain packing observed in β/α-barrels. The implication is that the 2 structural classes of β/α-barrel cores can arise divergently from an ancestral barrel framework and that convergent evolution to a stable fold need not be invoked to account for the emergence of 2 classes of β/α-barrel core.  相似文献   

6.
Integral membrane proteins come in two types, α-helical and β-barrel proteins. In both types, all hydrogen bonding donors and acceptors of the polypeptide backbone are completely compensated and buried while nonpolar side chains point to the membrane. The α-helical type is more abundant and occurs in cytoplasmic (or inner) membranes, whereas the β-barrels are known from outer membranes of bacteria. The β-barrel construction is described by the number of strands and the shear number, which is a measure for the inclination angle of the β-strands against the barrel axis. The common right-handed β-twist requires shear numbers slightly larger than the number of strands. Membrane protein β-barrels contain between 8 and 22 β-strands and have a simple topology that is probably enforced by the folding process. The smallest barrels form inverse micelles and work as enzymes or they bind to other macromolecules. The medium-range barrels form more or less specific pores for nutrient uptake, whereas the largest barrels occur in active Fe2+ transporters. The β-barrels are suitable objects for channel engineering, because the structures are simple and because many of these proteins can be produced into inclusion bodies and recovered therefrom in the exact native conformation.  相似文献   

7.
A multiple alignment of five (beta/alpha)8-barrel enzymes has been derived from their structure. The eight beta-strands and eight alpha-helices of the (beta/alpha)8-barrel are correctly aligned and the equivalenced residues in these regions fulfil similar structural roles. Each beta-strand has a central core of usually four residues, two residues contribute side-chains to the barrel core and the other two residues are involved in beta-strand/alpha-helix contacts. However, the fold imposes no constraints on the volumes of the residues at either a local or global level: the volume of the beta-barrel core varies between 1088 A3 in glycolate oxidase and 1571 A3 in taka-amylase. Sequence motifs derived from the multiple alignment were scanned against a database of 124 protein sequences, including 17 (beta/alpha)8-barrel enzymes. The results were evaluated in terms of the discrimination of (beta/alpha)8-barrel sequences and the quality of the alignments obtained. One motif was able to identify the top 12% of high scoring sequences as forming (beta/alpha)8-barrels with 50% accuracy and the bottom 50% of sequences as not being (beta/alpha)8-barrel proteins with 100% accuracy. However, in most instances the alignments were poor. The reasons for this are discussed with reference to the (beta/alpha)8-barrel proteins and the sequence motif method in general.  相似文献   

8.
The (betaalpha)(8)-barrel enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (tHisA) and imidazole glycerol phosphate synthase (tHisF) from Thermotoga maritima catalyze two successive reactions in the biosynthesis of histidine. In both enzymes, aspartate residues at the C-terminal end of beta-strand 1 (Asp8 in tHisA and Asp11 in tHisF) and beta-strand 5 (Asp127 in tHisA and Asp130 in tHisF) are essential for catalytic activity. It was demonstrated earlier that in tHisA the substitution of Asp127 by valine (tHisA-D127V) generates phosphoribosylanthranilate isomerase (TrpF) activity, a related (betaalpha)(8)-barrel enzyme participating in tryptophan biosynthesis. It is shown here that in tHisF the corresponding substitution of Asp130 by valine (tHisF-D130V) also generates TrpF activity. To determine the effectiveness of individual amino acid exchanges in these conversions, each of the 20 standard amino acid residues was introduced at position 127 of tHisA and 130 of tHisF by saturation random mutagenesis. The tHisA-D127X and tHisF-D130X variants with TrpF activity were identified by selection in vivo, and the proteins purified and characterized. The results obtained show that removal of the negatively charged carboxylate side-chain at the C-terminal end of beta-strand 5 is sufficient to establish TrpF activity in tHisA and tHisF, presumably because it allows the binding of the negatively charged TrpF substrate, phosphoribosylanthranilate. In contrast, the double mutants tHisA-D8N+D127V and tHisF-D11N+D130V did not show detectable activity, demonstrating that the aspartate residues at the C-terminal end of beta-strand 1 are essential for catalysis of the TrpF reaction. The ease with which TrpF activity can be established on both the tHisA and tHisF scaffolds supports the evolutionary relationship of these three enzymes and highlights the functional plasticity of the (betaalpha)(8)-barrel enzyme fold.  相似文献   

9.
Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed.  相似文献   

10.
It has been suggested that the common (betaalpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of identical (betaalpha)(4)-half barrels, followed by the optimisation of their interface. In our attempts to reconstruct these events in vitro we have previously linked in tandem two copies of the C-terminal half barrel HisF-C of imidazole glycerol phosphate synthase from Thermotoga maritima and subsequently reconstituted in the fusion construct HisF-CC a salt bridge cluster present in wild-type HisF. The resulting recombinant protein HisF-C*C, which was produced in an insoluble form and unfolded with low cooperativity at moderate urea concentrations has now been stabilised and solubilised by a combination of random mutagenesis and selection in vivo. For this purpose, Escherichia coli cells were transformed with a plasmid-based gene library encoding HisF-C*C variants fused to chloramphenicol acetyltransferase (CAT). Stable and soluble variants were identified by the survival of host cells on solid medium containing high concentrations of the antibiotic. The selected HisF-C*C proteins, which were characterised in vitro in the absence of CAT, contained eight different amino acid substitutions. One of the exchanges (Y143C) stabilised HisF-C*C by the formation of an intermolecular disulfide bond. Three of the substitutions (G245R, V248M, L250Q) were located in the long loop connecting the two HisF-C copies, whose subsequent truncation from 13 to 5 residues yielded the stabilised variant HisF-C*C Delta. From the remaining substitutions, Y143H and V234M were most beneficial, and molecular dynamics simulations suggest that they strengthen the interactions between the half barrels by establishing a hydrogen-bonding network and an extensive hydrophobic cluster, respectively. By combining the loop deletion of HisF-C*C Delta with the Y143H and V234M substitutions, the variant HisF-C**C was generated. Recombinant HisF-C**C is produced in soluble form, forms a pure monomer with its tryptophan residues shielded from solvent and unfolds with similar cooperativity as HisF. Our results show that, starting from two identical and fused half barrels, few amino acid exchanges are sufficient to generate a highly stable and compact (betaalpha)(8)-barrel protein with wild-type like structural properties.  相似文献   

11.
The (beta/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.  相似文献   

12.
Most proteins found in the outer membrane of gram-negative bacteria share a common domain: the transmembrane β-barrel. These outer membrane β-barrels (OMBBs) occur in multiple sizes and different families with a wide range of functions evolved independently by amplification from a pool of homologous ancestral ββ-hairpins. This is part of the reason why predicting their three-dimensional (3D) structure, especially by homology modeling, is a major challenge. Recently, DeepMind's AlphaFold v2 (AF2) became the first structure prediction method to reach close-to-experimental atomic accuracy in CASP even for difficult targets. However, membrane proteins, especially OMBBs, were not abundant during their training, raising the question of how accurate the predictions are for these families. In this study, we assessed the performance of AF2 in the prediction of OMBBs and OMBB-like folds of various topologies using an in-house-developed tool for the analysis of OMBB 3D structures, and barrOs. In agreement with previous studies on other membrane protein classes, our results indicate that AF2 predicts transmembrane β-barrel structures at high accuracy independently of the use of templates, even for novel topologies absent from the training set. These results provide confidence on the models generated by AF2 and open the door to the structural elucidation of novel transmembrane β-barrel topologies identified in high-throughput OMBB annotation studies or designed de novo.  相似文献   

13.
The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane β-barrel proteins. These β-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane β-barrels include that of a full length autotransporter (EstA), a bacterial heme transporter complex (HasR), a bacterial porin in complex with several ligands (PorB), and the mitochondrial voltage-dependent anion channel (VDAC) from both mouse and human. These represent only a few of the interesting structures of β-barrel membrane proteins recently elucidated. However, they demonstrate many of the advancements made within the field of transmembrane protein structure in the past few years.  相似文献   

14.
Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPSDelta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension.  相似文献   

15.
The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.  相似文献   

16.
The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively β-barrels composed of between 8 and 26 β-strands. To explore the relationship between β-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 β-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the β-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in β-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in β-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.  相似文献   

17.
The present review describes the structural features of alpha-amylase, beta-amylase and glucoamylase that are the best known amylolytic enzymes. Although they show similar function, i.e. catalysis of hydrolysis of alpha-glucosidic bonds in starch and related saccharides, they are quite different. alpha-Amylase is the alpha --> alpha retaining glycosidase (it uses the retaining mechanism), and beta-amylase together with glucoamylase are the alpha --> beta inverting glycosidases (they use the inverting mechanism). While beta-amylase and glucoamylase form their own families 14 and 15, respectively, in the sequence-based classification of glycoside hydrolases, alpha-amylase belongs to a large clan of three families 13, 70 and 77 consisting of almost 30 different specificities. Structurally both alpha-amylase and beta-amylase rank among the parallel (beta/alpha)8-barrel enzymes, glucoamylase adopts the helical (alpha/alpha)6-barrel fold. The catalytic (beta/alpha)8-barrels of alpha-amylase and beta-amylase differ from each other. The only common sequence-structural feature is the presence of the starch-binding domain responsible for the binding and ability to digest raw starch. It is, however, present in about 10% of amylases and behaves as an independent evolutionary module. A brief discussion on structure-function and structure-stability relationships of alpha-amylases and related enzymes is also provided.  相似文献   

18.
The (beta(alpha))8-barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (beta(alpha))8-barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active-site-forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG-PRAI could be identified from a 10(6)-fold excess of a FLAG-negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG-PRAI loop library of approximately 10(7) clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (beta(alpha))8-barrel consisting of (beta(alpha))1-5beta6. Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21-fold increase in mAb M2 affinity compared with full-length FLAG-PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (beta(alpha))8-barrel fold.  相似文献   

19.
The aim of this study was to increase the stability of the thermolabile (betaalpha)8-barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges. For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines. The variant linking residues 3 and 189 fastens the N-terminus to the (betaalpha)8-barrel. The rate of thermal inactivation at 50 degrees C of this variant with a closed disulfide bridge is 65-fold slower than that of the reference dithiol form, but only 13-fold slower than that of the parental protein. The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified. To confirm these data, we have solved the X-ray structure to 2.1-A resolution. The second variant was designed to crosslink the terminal modules betaalpha1 and betaalpha8. However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein.  相似文献   

20.

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the 13C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The 13C chemical shifts are shown in 2D 13C–13C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The 13C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

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