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1.
A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic distribution of the mutant virus was delayed by 5-6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus. 相似文献
2.
Porin (341 amino acids; M(r) 37 782) of Haemophilus influenzae type b mediates exchange of solutes between the external environment and the periplasm of this Gram-negative bacterium. Positively charged residues in the extracellular loops have been shown to be involved in the voltage gating of this protein. To further elucidate our observations on the functional properties of this channel, we mutated seven lysines (Lys(48), Lys(161), Lys(165), Lys(170), Lys(248), Lys(250), and Lys(253)) to glutamic acid. The selected residues were previously shown to be accessible to chemical modification, and they map to three locations: loop 4 and loop 6, and within the barrel lumen. The seven mutant proteins were purified, and each was reconstituted into planar lipid bilayers to characterize its channel forming properties. The single substitution mutant porins displayed increased single channel conductances in 1 M KCl ranging between 134 and 178% of the single channel conductance for wild-type Hib porin. Six of the seven mutant porins also displayed altered current-voltage relationships when compared to wild-type Hib porin. Whereas Lys(170)Glu had activity similar to wild-type Hib porin, Lys(48)Glu, Lys(248)Glu, and Lys(253)Glu showed substantial voltage gating at both positive and negative polarities. Lys(161)Glu and Lys(250)Glu gated only at negative potentials, and Lys(165)Glu gated only at positive potentials. Rather than ascribing one specific loop in gating, our analyses of these mutant Hib porins suggest that voltage gating can be attributed to contributions from loops 4 and 6 and a residue within the barrel lumen. 相似文献
3.
A study was made of the coat protein (CP) of thermosensitive semidefective tobacco mosaic virus strain K1 (TMV-K1). In contrast
to CP of other TMV strains, K1 CP showed high nonspecific aggregation and did not form normal two-layered cylindrical aggregates.
In none of the conditions tested, K1 CP formed virions with cognate K1 RNAin vitro. The abnormal properties were attributed to substitution Lys53→Glu differentiating the K1 CP from those of other tobamoviruses.
It is assumed that the high structural plasticity allows the tobamovirus virions to incorporate CP subunits even with unfavorable
amino acid changes. 相似文献
4.
A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin 总被引:14,自引:0,他引:14
R R Franke T P Sakmar D D Oprian H G Khorana 《The Journal of biological chemistry》1988,263(5):2119-2122
In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive. 相似文献
5.
Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process. 总被引:1,自引:0,他引:1
M R Ciriolo A Battistoni M Falconi G Filomeni G Rotilio 《European journal of biochemistry》2001,268(3):737-742
Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger. 相似文献
6.
K Yutani K Ogasahara K Aoki T Kakuno Y Sugino 《The Journal of biological chemistry》1984,259(22):14076-14081
To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized. 相似文献
7.
Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa
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The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling. 相似文献
8.
We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%). 相似文献
9.
Matsuura Y Takehira M Sawano M Ogasahara K Tanaka T Yamamoto H Kunishima N Katoh E Yutani K 《The FEBS journal》2012,279(1):78-90
The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C. 相似文献
10.
R Geiger T Rüter J Alves A Fliess H Wolfes V Pingoud C Urbanke G Maass A Pingoud A Düsterh?ft 《Biochemistry》1989,28(6):2667-2677
We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)]. 相似文献
11.
MacGillivray RT Bewley MC Smith CA He QY Mason AB Woodworth RC Baker EN 《Biochemistry》2000,39(6):1211-1216
Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site. 相似文献
12.
D P Sun E S?derlind W A Baase J A Wozniak U Sauer B W Matthews 《Journal of molecular biology》1991,221(3):873-887
Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide
bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals
that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in
other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive
site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only
4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other
Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating
that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89)
is not required. 相似文献
14.
Kono M 《FEBS letters》2006,580(1):229-232
Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation. 相似文献
15.
Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gαi) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gαi. On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life. 相似文献
16.
Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer. 总被引:2,自引:0,他引:2
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C. Z. Ni C. A. White R. S. Mitchell J. Wickersham R. Kodandapani D. S. Peabody K. R. Ely 《Protein science : a publication of the Protein Society》1996,5(12):2485-2493
There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine. 相似文献
17.
The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo.
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The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo. 相似文献
18.
Cindy L. Fisher Diane E. Cabelli John A. Tainer Robert A. Hallewell Elizabeth D. Getzoff 《Proteins》1994,19(1):24-34
Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc. 相似文献
19.
Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds 总被引:1,自引:0,他引:1
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To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained approximately 12-fold or approximately 5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic approximately 80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to approximately 38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism. 相似文献
20.
Although the biological variability of Watermelon mosaic virus is limited, isolates from the three main molecular groups differ in their ability to infect systemically Chenopodium quinoa. Mutations were introduced in a motif of three or five amino acids located in the N‐terminal part of the coat protein, and differing in isolates from group 1 (motif: lysine‐glutamic acid‐alanine (Lys‐Glu‐Ala) or KEA, systemic on C. quinoa), group 2 (Lys‐Glu‐Thr or KET, not systemic on C. quinoa) and group 3 (KEKET, not systemic on C. quinoa). Mutagenesis of KEKET in an isolate from group 3 to KEA or KEKEA was sufficient to make the virus systemic on C. quinoa, whereas mutagenesis to KET had no effect. Introduction of a KEA motif in Zucchini yellow mosaic virus coat protein also resulted in systemic infection on C. quinoa. These mutations had no obvious effect on the disorder profile or potential post‐translational modifications of the coat protein as determined in silico. 相似文献