scholarly journals Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.

1994 ◽  
Vol 14 (1) ◽  
pp. 815-821 ◽  
Author(s):  
P Poullet ◽  
B Lin ◽  
K Esson ◽  
F Tamanoi
Keyword(s):  
Amino Acids ◽  
Neurofibromatosis Type ◽  
Type I ◽  
Ras Proteins ◽  
Wild Type ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
Quantitative Analyses ◽  
Intrinsic Gtpase Activity

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.

Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes

1994 ◽  
Vol 14 (1) ◽  
pp. 815-821
Author(s):  
P Poullet ◽  
B Lin ◽  
K Esson ◽  
F Tamanoi
Keyword(s):  
Amino Acids ◽  
Neurofibromatosis Type ◽  
Type I ◽  
Ras Proteins ◽  
Wild Type ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
Quantitative Analyses ◽  
Intrinsic Gtpase Activity

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.

scholarly journals The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 926
Author(s):  
Maria C. Martins ◽  
Susana F. Fernandes ◽  
Bruno A. Salgueiro ◽  
Jéssica C. Soares ◽  
Célia V. Romão ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Reductase Activity ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Bona Fide ◽  
Oxygen Reductase ◽  
Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

scholarly journals Analysis of Genomes Changes in Pseudomonas Chlororaphis Subsp. Aurantiaca Strains Producing Phenazines

2021 ◽  
Author(s):  
Anastasia I. Liaudanskaya ◽  
Natalia P. Maximova ◽  
Katsiaryna G. Verameyenka
Keyword(s):  
Mutant Strain ◽  
Phenylalanine Hydroxylase ◽  
Response Regulator ◽  
Trna Genes ◽  
Type I ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Domain Structures ◽  
Genome Annotations

Abstract Genomes of three strains – phenazine producers – Pseudomonas chlororaphis subsp. aurantiaca (B-162 (wild-type), mutant strain B-162/255 and its derivatives B-162/17) were sequenced and compared. All genome annotations revealed 6347 CDS, 5 rRNA clusters (5S, 16S, 23S) and 59 tRNA genes. Comparison analysis of wild-type strain and B-162/255 mutant strain genomes allowed revealing 32 mutations. 19 new mutations were detected upon comparison of genomes strains B-162/255 and B-162/17. Further bioinformatics analysis allowed predicting mutant proteins` functions and secondary structures of five gene products, mutations in which might potentially have influence on phenazine synthesis and secretion in Pseudomonas bacteria. These genes are phenylalanine hydroxylase transcriptional activator PhhR, type I secretion system ATPase, transcriptional regulator MvaT, GacA response regulator and histidine kinase. Amino acid substitutions were located in domain structures of corresponding proteins.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

Tumor Suppressor p16INK4A:  Structural Characterization of Wild-Type and Mutant Proteins by NMR and Circular Dichroism†

Biochemistry ◽  
1996 ◽  
Vol 35 (29) ◽  
pp. 9475-9487 ◽  
Author(s):  
Anton Tevelev ◽  
In-Ja L. Byeon ◽  
Thomas Selby ◽  
Karen Ericson ◽  
Hee-Jung Kim ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Tumor Suppressor ◽  
Structural Characterization ◽  
Wild Type ◽  
Mutant Proteins ◽  
Circular Dichroïsm

Molecular characterization of MHC class II DRA cDNA in Deoni cattle

2018 ◽  
Author(s):  
K. Swathi ◽  
M. Gnana Prakash ◽  
D. Sakaram ◽  
T. Raghunandan ◽  
A. Sarat Chandra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mhc Class Ii ◽  
Signal Peptide ◽  
Class Ii ◽  
Glycosylation Site ◽  
Amino Acid Sequences ◽  
Conserved Residues ◽  
A2 Domain

The present study was undertaken to clone and characterize DRA gene in Deoni cattle. The cDNA for the DRA gene was amplified by using specific primers designed based on available cattle sequences and purified products were cloned in competent E.coli (DH5á) strain. The full length 1013bp product of cDNA of DRA contained a single ORF of 762 nucleotides that coded for 253 amino acids translated product. Twenty four amino acids formed signal peptide while 229 constituted mature peptide. The deduced amino acid sequences resembled those of class II molecules of other species for all the conserved residues having critical functional role. But a single N-linked glycosylation site in á1 was observed in cattle and buffalo when compared to human and swine which contain a second site in á2 domain. The signal peptide was found more variable among the species compared. Comparison of nucleotide and amino acid sequences among related species and dendrogram constructed revealed that the cattle sequences are more similar to buffalo sequences.

Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides

2009 ◽  
Vol 50 (6) ◽  
pp. 483-492 ◽  
Author(s):  
ZHENGXIN WANG ◽  
WALTER P. CARNEY ◽  
RICHARD A. LAURSEN
Keyword(s):  
Ras Proteins ◽  
Wild Type ◽  
Membrane Bound ◽  
Human Wild Type
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