Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.
J Mol Biol 1992 May 20
PMID:Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation. 159 23

The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in the trp A gene. Screening of the resulting clones allowed selection of non-interactive mutant alpha subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: alpha 126 valine (GTG)----glutamic acid (GAG) and alpha 128 valine (GTT)----aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as alpha 66 asparagine (AAC)----aspartic acid (GAC); alpha 109 lysine (AAA)----arginine (AGA); alpha 118 cysteine (TGC)----arginine (CGC). Where possible, we individually assessed the importance of these residues in alpha beta interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.
Mol Gen Genet 1992 May
PMID:Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit. 160 55

The periplasmic binding protein LAO from Salmonella typhimurium, which is involved in lysine, arginine and ornithine transport, has been crystallized together with one of its ligands, arginine (LAO-Arg). Preliminary X-ray diffraction studies of LAO-Arg crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1) and has the unit cell dimensions of a = 37.65 A, b = 59.45 A, c = 115.91 A. Crystals of the LAO-Arg complex diffract beyond 2.0 A resolution.
J Mol Biol 1992 Jun 20
PMID:Crystallization and preliminary X-ray studies of the liganded lysine, arginine, ornithine-binding protein from Salmonella typhimurium. 161 94

A model of the Tetrahymena catalytic site predicts that nucleotide 262 (nt262) caps an RNA pocket in which nucleoside substrates and arginine-like competitive inhibitors reside. Here we show that substituted RNAs behave as if nt262 stacks on nucleoside substrates, supporting the model. The more frequent an nt262 is in natural sequences, the more reactive the corresponding Tetrahymena RNA is for both cognate and non-cognate nucleoside substrates. These more reactive RNAs with the majority nt262 also bind arginine more strongly, stereoselect more strongly in favor of L-arginine, and make a greater distinction between the somewhat similar side-chains of L-arginine and L-lysine. These parallel changes in interaction with nucleosides and arginine analogs seem best explained by stacking of the arginine's guanidino group under the nt262 base. One consequence is that selection for improved Tetrahymena catalysis with nucleosides should also yield an improved arginine site.
J Mol Biol 1992 Jun 20
PMID:Co-optimization of ribozyme substrate stacking and L-arginine binding. 161

Translation initiation factor eIF-5A is an abundant protein in which a lysine residue is modified by spermidine to form the amino acid derivative, hypusine. The factor is encoded by two genes in Saccharomyces cerevisiae, called TIF51A and TIF51B, which are regulated reciprocally by oxygen and by heme. TIF51B, also called ANB1, is located on chromosome X in a region called COR. We physically mapped TIF51A and its associated serine tRNA2 gene by the method of chromosome fragmentation and pulsed-field gel electrophoresis. TIF51A maps 90 kb from the left end of chromosome V in a region called ARC. The COR and ARC regions contain CYC1 and CYC7, respectively, and appear to be duplications carrying numerous related genes. The arrangements of related genes in the two regions are incompatible with a duplication mechanism involving a circular intermediate.
Mol Gen Genet 1992 Jun
PMID:The two genes encoding protein synthesis initiation factor eIF-5A in Saccharomyces cerevisiae are members of a duplicated gene cluster. 162 Jan 3

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.
Plant Mol Biol 1992 Aug
PMID:Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts. 164 84

The diaminopimelate (DAP) pathway provides the cell with lysine and with DAP, a vital cell wall constituent. Mutations in the DAP pathway of lysine biosynthesis are lethal for cells exposed to lysine in the absence of DAP. In this paper, the substitution of the dapD gene of Escherichia coli with the kanamycin resistance gene from Tn903 is described and its possible uses are discussed.
Mol Gen Genet 1991 May
PMID:Development of stable, genetically well-defined conditionally viable Escherichia coli strains. 164 86

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.
J Mol Biol 1991 Aug 05
PMID:Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution. 165 96

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.
Mol Endocrinol 1991 Mar
PMID:Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin. 165 94

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.
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PMID:Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase. 165 23


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