共查询到20条相似文献,搜索用时 15 毫秒
1.
Roberge Martin; Shareck Franciois; Morosoli Rolf; Kluepfel Dieter; Dupont Claude 《Protein engineering, design & selection : PEDS》1999,12(3):251-257
The role of four aromatic residues (W85, Y172, W266 and W274)in the structurefunction relationship in xylanase A fromStreptomyces lividans (XlnA) was investigated by site-directedmutagenesis where each residue was subjected to three substitutions(W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four aminoacids are highly conserved among family 10 xylanases and structuraldata have implicated them in substrate binding at the activesite. Far-UV circular dichroism spectroscopy was used to showthat the overall structure of XlnA was not affected by any ofthese mutations. High-performance liquid chromatographic analysisof the hydrolysis products of birchwood xylan and xylopentaoseshowed that mutation of these aromatic residues did not alterthe enzyme's mode of action. As expected, though, it did reducethe affinity of XlnA for birchwood xylan. A comparison of thekinetic parameters of different mutants at the same positiondemonstrated the importance of the aromatic nature of W85, Y172and W274 in substrate binding. Replacement of these residuesby a phenylalanine resulted in mutant proteins with a KM closerto that of the wild-type protein in comparison with the othermutations analyzed. The kinetic analysis of the mutant proteinsat position W266 indicated that this amino acid is importantfor both substrate binding and efficient catalysis by XlnA.These studies also demonstrated the crucial role of these activesite aromatic residues for the thermal stability of XlnA. 相似文献
2.
Roberge M; Dupont C; Morosoli R; Shareck F; Kluepfel D 《Protein engineering, design & selection : PEDS》1997,10(4):399-403
Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA)
from Streptomyces lividans, belonging to family 10 and superfamily 4/7 of
glycosyl hydrolases, was chosen to study the role of this conserved
residue. The isosteric mutation N127D introduced did not affect the fold of
XlnA as revealed by circular dichroism. Comparison of the kinetic constants
of N127D and wild-type XlnA revealed a 70-fold decrease in the specificity
constant (kcat/K(M)) towards birchwood xylan, which is attributed solely to
the difference in the kcat value and indicates a role of N127 in
stabilization of the catalytic intermediate. N127 also plays a role in
maintaining the ionization states of the two catalytic residues, as shown
by the modified pH profile of XlnA-N127D. Characterization of XlnA-N127D
and the analysis of the three-dimensional structure of XlnA converge
towards a stabilization role for N127 in the catalytic site of XlnA.
相似文献
3.
Chiti F; Magherini F; Taddei N; Ilardi C; Stefani M; Bucciantini M; Dobson CM; Ramponi G 《Protein engineering, design & selection : PEDS》1998,11(7):557-561
An oligonucleotide-directed mutagenesis study was carried out on the five
acylphosphatase conserved lysine residues to assess their possible
participation in enzyme active site formation and their contribution to the
enzyme conformational stability. The study was designed to eliminate the
ambiguity arising from the presence of a sulfate ion, an enzyme competitive
inhibitor, bound to lysine 32 and 68 in the crystal structure of the
erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the
presence of residues with pKa=7.9 and 11, tentatively identified as two
lysines. The kinetic parameters for the mutants under investigation are not
significantly different from those of the wild-type enzyme, demonstrating
that none of the lysine residues are involved in catalysis or in substrate
binding. In addition, thermal and urea denaturation experiments performed
by circular dichroism indicate that the mutated lysine residues do not play
a significant role in the enzyme structural stabilization, as the
destabilizing energy averages 1.40 kJ/mol. Such results are in agreement
with those obtained with other proteins indicating that lysine residues
make little contribution to the stability of the native structure.
相似文献
4.
In the TNC family of Ca-binding proteins (calmodulin, parvalbumin,intestinal calcium binding protein and troponin C) {small tilde}70 well-conserved amino acid sequences and six crystal structuresare known. We find a clear correlation between residue contactsin the structures and residue conservation in the sequences:residues with strong sidechainsidechain contacts in thethree-dimensional structure tend to be the more conserved inthe sequence. This is one way to quantify the intuitive notionof the importance of sidechain interactions for maintainingprotein three-dimensional structure in evolution and may usefullybe taken into account in planning point mutations in proteinengineering. 相似文献
5.
The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMPdependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solventexposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR). 相似文献
6.
Vincenzetti S.; Cambi A.; Maury G.; Bertorelle F.; Gaubert G.; Neuhard J.; Natalini P.; Salvatori D.; De Sanctis G.; Vita A. 《Protein engineering, design & selection : PEDS》2000,13(11):791-799
Site-directed mutagenesis on human cytidine deaminase (CDA)was employed to mutate specifically two highly conserved phenylalanineresidues, F36 and F137, to tryptophan; at the same time, theunique tryptophan residue present in the sequence at position113 was mutated to phenylalanine. These double mutations wereperformed in order to have for each protein a single tryptophansignal for fluorescence studies relative to position 36 or 137.The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F,showed by circular dicroism and thermal stability an overallstructure not greatly affected by the mutations. The titrationof Trp residues by N-bromosuccinimide (NBS) suggested that residueW113 of the wild-type CDA and W36 of mutant F36W/W113F are buriedin the tertiary structure of the enzyme, whereas the residueW137 of mutant F137W/W113F is located near the surface of themolecule. Kinetic experiments and equilibrium experiments withFZEB showed that the residue W113 seems not to be part of theactive site of the enzyme whereas the Phe/Trp substitution inF36W/W113F and F137W/W113F mutant enzymes had a negative effecton substrate binding and catalysis, suggesting that F137 andF36 of the wild-type CDA are involved in a stabilizing interactionbetween ligand and enzyme. 相似文献
7.
Ortiz-Soto ME Rivera M Rudiño-Piñera E Olvera C López-Munguía A 《Protein engineering, design & selection : PEDS》2008,21(10):589-595
Levansucrases (LS) are fructosyltransferases (FTFs) belonging to family 68 of glycoside hydrolases (GH68) using sucrose as substrate to synthesize levan, a fructose polymer. From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated. A product specificity modification was obtained with mutants Y429N and R433A that no longer produce levan but exclusively oligosaccharides. An effect of the mutation S164A was observed on enzyme stability and kinetic behavior; this mutation also induces a levan activation effect that enhances the reaction rate. We report the crystallographic structure of this mutant and found that S164 is an important residue to maintain the nucleophile position in the active site. We also found evidence of the important role of Y429 in acceptor specificity: this is a key residue coordinating the sucrose position in the catalytic domain-binding pocket. Some of these mutations resulted in LS with a broad range of specificities and new biochemical properties. 相似文献
8.
Evidence for a second conserved arginine residue in the integrase family of recombination proteins 总被引:10,自引:0,他引:10
Abremski Kenneth E.; Hoess Ronald H. 《Protein engineering, design & selection : PEDS》1992,5(1):87-91
This study was designed to search for new regions of similarityin the integrase family of recombination proteins which consistsof 28 members found in bacteria and yeast. A computer methodbased on an information content analysis has been used to alignlocal regions of homology in the set of unaligned protein sequencesfrom this family. Among the aligned regions with high informationcontent were those containing the known conserved histidine,arginine and tyrosine residues. In addition, a new region wasidentified containing another arginine residue that appearsto be conserved in all members of the family. To test furtherthe importance of this newly identified arginine residue, mutantsin the Cre protein from phage PI, a member of this integrasefamily, have been constructed which alter this residue. Themutations which change arginine to lysine and arginine to cysteinedepress catalytic activity but not site-specific binding tothe lox site. This result is expected for a conserved activesite residue. This computer analysis also provides a means forsearching for new members of the integrase family 相似文献
9.
The deficiency of human carbonic anhydrase II (HCAII) has been recognized to be associated with a disease called CAII deficiency syndrome (CADS). Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding. However, sequence alignment indicated that the 237th residue of CAII is not fully conserved across all species. The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol). The experimental determination indicated that at least three mutations affect HCAII folding significantly and the P237H mutation was the most deleterious one, suggesting that Pro237 was important to HCAII folding. The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations. 相似文献
10.
Kornhaber GJ Tropak MB Maegawa GH Tuske SJ Coales SJ Mahuran DJ Hamuro Y 《Chembiochem : a European journal of chemical biology》2008,9(16):2643-2649
Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions. 相似文献
11.
Antibody 1E9, which binds a tetrachloronorbornene derivative with subnanomolar affinity and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, arose from a family of highly restricted germ-line immunoglobulins that bind diverse hydrophobic ligands. Two somatic mutations, one at position L89 in the light chain (SerL89Phe) and another at position H47 in the heavy chain (TrpH47Leu), have been postulated to be responsible for the unusually high degree of shape and chemical complementarity observed in the crystal structure of 1E9 complexed with its hapten. To test this hypothesis, the germ-line sequence at these two positions was restored by site-directed mutagenesis. The ensuing 160 to 3900-fold decrease in hapten affinity and the complete loss of catalytic activity support the hypothesis that these somatic mutations substantially remodel the antibody binding pocket. Mutation of the highly conserved hydrogen-bond donor AsnH35, which sits at the bottom of the active site and is a hallmark of this family of antibodies, is also catastrophic with respect to hapten binding and catalysis. In contrast, residues in the CDR H3 loop, which contributes a significant fraction of the hapten-contacting protein surface, have a more subtle influence on the properties of 1E9. Interestingly, while most changes in this loop have neutral or modestly deleterious effects, replacement of MetH100b at the floor of the pocket with phenylalanine leads to a significant sevenfold increase in catalytic activity. The latter result is surprising given the unusually close fit of the parent antibody to the transition-state analogue. Further fine-tuning of the interactions between 1E9 and its ligands by introducing mutations outside the active site could conceivably yield substantially more active catalysts. 相似文献
12.
Vázquez-Figueroa E Chaparro-Riggers J Bommarius AS 《Chembiochem : a European journal of chemical biology》2007,8(18):2295-2301
Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing the widespread adoption of biocatalysts for industrial synthesis. A crucial distinction of many redox enzymes used to synthesize chiral compounds is the need for cofactors (e.g., NAD(P)(H)) for function. Because of the prohibitively high prices of nicotinamide cofactors, a robust cofactor-regenerating enzyme is required for the economical synthesis of fine chemicals by biocatalysis. Here we test the structure-guided consensus for the generation of a thermostable glucose dehydrogenase (GDH). The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46 %. Of the 24 variants, seven were located at the subunit interface-known to influence GDH stability-and six were more stable (86 % success). The best variants feature a half-life of approximately 3.5 days at 65 degrees C, in contrast to approximately 20 min at 25 degrees C for the wild type, thus enhancing stability 10(6)-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologues from Bacillus thuringiensis and Bacillus licheniformis. The thermal stability as measured by half-life and CD(222 nm) of the GDH variants was increased, as expected. The resulting stability changes provide further support for the view that these residues are critical for stability of GDHs and reinforce the success of the consensus approach for identifying stabilizing mutations. 相似文献
13.
Fisher Z Boone CD Biswas SM Venkatakrishnan B Aggarwal M Tu C Agbandje-McKenna M Silverman D McKenna R 《Protein engineering, design & selection : PEDS》2012,25(7):347-355
Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties. 相似文献
14.
Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes 总被引:1,自引:0,他引:1
Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-
Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the
N-terminus of the enzyme is located at or near the enzyme's active center
involved in Mn(II)-L-malate binding and is also near to the subunits'
interface. In the present study, the conformational stability of the
various deletion (delta) and substitution mutants at Lys2/Lys3 of the
enzyme was investigated with chemical and thermal sensitivities. The lysine
residue at position 2 or 3 seems to be crucial for the correct active site
conformation, probably through an ion-pairing with Glu6. Deletion at Lys2
or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable
than the wild-type enzyme towards chemical denaturation. Kinetic analysis
of the thermal inactivation at 58 degrees C of the recombinant enzymes
indicated that mutation at position 3 to alanine (K3A) endows the protein
with extra stability compared with the wild-type enzyme. K3A is also stable
towards chemical denaturation. The concentration of urea that causes half
unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the
wild-type enzyme. The K3A mutant of malic enzyme might therefore have
potential practical applications.
相似文献
15.
Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase 总被引:1,自引:0,他引:1
The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR)
has been explored by a series of insertion and deletion mutations.
Insertion of a seven amino acid sequence from a structurally equivalent
'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6
kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34- fold. Deletion
of F137, delta 137, the looped out residue in the bulge, also destabilizes
E. coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency
threefold. Concurrent deletion of F137 and mutation of, V136 to proline to
try and maintain the strand twist associated with the beta-bulge decreases
protein stability by 3.4 kcal/mol and decreases catalytic efficiency
84-fold. These insertion/deletion mutations were also constructed in a D27S
DHFR background. The D27S mutation has been described previously and
proposed to remove the catalytic acid from the active site. The delta 137
mutation partially suppresses the effect of the D27S mutation as it
decreases the K(m) for substrate, dihydrofolate, twofold. Non-additive
effects are observed for the insertion/deletion mutations in wild-type
versus D27S DHFR backgrounds, consistent with structural changes.
相似文献
16.
Koutsioulis D Wang E Tzanodaskalaki M Nikiforaki D Deli A Feller G Heikinheimo P Bouriotis V 《Protein engineering, design & selection : PEDS》2008,21(5):319-327
Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E. 相似文献
17.
研究了meso-四卟啉和meso-四卟啉的离解常数。TBPhP的离解常数为:pKa4=1.13.pKa3=1.88,pKA2=12.21;TTPS的离解常数为:pKa4=4.47,pKa3=6.26。外还发现了两卟啉的酸碱诱导聚合。 相似文献
18.
L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad
substrate specificity and utilizes a wide range of 2-oxo acids branched at
the C4 atom. Modifications have been made to the sequence of the
NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and
alter the region of substrate specificity towards various 2- oxocarbonic
acids. All variations were based on a 3D-structure model of the enzyme
using the X-ray coordinates of the functionally related L- lactate
dehydrogenase (L-LDH) from dogfish as a template. This protein displays
only 23% sequence identity to L-HicDH. The active site of L- HicDH was
modelled by homology to the L-LDH based on the conservation of
catalytically essential residues. Substitutions of the active site residues
Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify
their unique participation in substrate recognition and orientation. The
kinetic properties of the L239A, L239M, L236V and T245A enzyme variants
confirmed the structural model of the active site of L-HicDH. The
substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate,
phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the
active site of the subsequently refined model. In order to design
dehydrogenases with an improved substrate specificity towards keto acids
branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236
and Thr245 were introduced and resulted in mutant enzymes with completely
different substrate specificities. The substitution T245A resulted in a
relative shift of substrate specificity for keto-tert-leucine of more than
17000 compared with the 2-oxocaproate (kcat/KM). For the substrates
branched at C4 a relative shift of up to 500 was obtained for several
enzyme variants. A total of nine mutations were introduced and the kinetic
data for the set of six substrates were determined for each of the
resulting mutant enzymes. These were compared with those of the wild-type
enzyme and rationalized by the active site model of L-HicDH. An analysis of
the enzyme variants provided new insight into the residues involved in
substrate binding and residues of importance for the differences between
LDHs and HicDH. After the protein design project was complete the X-ray
structure of the enzyme was solved in our group. A comparison between the
model and the experimental 3D structure proved the quality of the model.
All the variants were designed, expressed and tested before the 3D
structure became available.
相似文献
19.
Tang Dahai; Ando Shoji; Takasaki Yozo; Tadano Jutaro 《Protein engineering, design & selection : PEDS》2000,13(4):283-289
We have performed mutational analyses of restriction endonucleaseHindIII in order to identify the amino acid residues responsiblefor enzyme activity. Four of the seven HindIII mutants, whichhad His-tag sequences at the N-termini, were expressed in Escherichiacoli, and purified to homogeneity. The His-tag sequence didnot affect enzyme activity, whereas it hindered binding of theDNA probe in gel retardation assays. A mutant E86K in whichLys was substituted for Glu at residue 86 exhibited high endonucleaseactivity. Gel retardation assays showed high affinity of thismutant to the DNA probe. Surprisingly, in the presence of atransition metal, Mo2+ or Mn2+, the E86K mutant cleaved substrateDNA at a site other than HindIII. Substitution of Glu for Valat residue 106 (V106E), and Asn for Lys at residue 125 (K125N)resulted in a decrease in both endonucleolytic and DNA bindingactivities of the enzyme. Furthermore, substitution of Leu forAsp at residue 108 (D108L) abolished both HindIII endonucleaseand DNA binding activities. CD spectra of the wild type andthe two mutants, E86K and D108L, were similar to each other,suggesting that there was little change in conformation as aresult of the mutations. These results account for the notionthat Asp108 could be directly involved in HindIII catalyticfunction, and that the substitution at residue 86 may bringabout new interactions between DNA and cations. 相似文献
20.
Fushinobu S; Ito K; Konno M; Wakagi T; Matsuzawa H 《Protein engineering, design & selection : PEDS》1998,11(12):1121-1128
Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable
at pH 1.0. The crystal structure of xylanase C was determined at 2.0 A
resolution (R-factor = 19.4%). The overall structure was similar to those
of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are
located at a hydrogen-bonding distance (2.8 A), as in other xylanases with
low pH optima. Asp37 of xylanase C was replaced with asparagine and other
residues by site-directed mutagenesis. Analyses of the wild-type and mutant
enzymes showed that Asp37 is important for high enzyme activity at low pH.
In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the
maximum specific activity decreased to about 15% of that of the wild-type
enzyme. On structural comparison with xylanases with higher pH optima,
another striking feature of the xylanase C structure was found; the enzyme
has numerous acidic residues concentrated on the surface (so-called
'Ser/Thr surface' in most family 11 xylanases). The relationship of the
stability against extreme pH conditions and high salt concentrations with
the spacially biased distribution of charged residues on the proteins is
discussed.
相似文献