scholarly journals Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii

1999 ◽  
Vol 344 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Jae Hyung AN ◽  
Gha Young LEE ◽  
Jin-Won JUNG ◽  
Weontae LEE ◽  
Yu Sam KIM
Keyword(s):  
Enzyme Catalysis ◽  
Hplc Analysis ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Database Analysis ◽  
Identify Amino Acid ◽  
Malonyl Coa

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either.

scholarly journals Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis

1996 ◽  
Vol 318 (3) ◽  
pp. 1041-1049 ◽  
Author(s):  
Himakshi PATEL ◽  
Jo BRAMALL ◽  
Helen WATERS ◽  
Maria C. DE BEER ◽  
Patricia WOO
Keyword(s):  
Amino Acid ◽  
Serum Amyloid A ◽  
Amyloid Fibrils ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Amyloid A ◽  
Amyloid Fibril Formation

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.3, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.

Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1

2002 ◽  
Vol 363 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Nerino ALLOCATI ◽  
Michele MASULLI ◽  
Enrico CASALONE ◽  
Silvia SANTUCCI ◽  
Bartolo FAVALORO ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cd Spectra ◽  
Glutathione S Transferase ◽  
Terminal Amino

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65→Asp and Glu65→Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103→Ala and Glu104→Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis.

scholarly journals Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Lina Kaminski ◽  
Jerry Eichler
Keyword(s):  
Biochemical Characterization ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Natural Substrate ◽  
Identify Amino Acid ◽  
Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

scholarly journals Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7804-7813 ◽  
Author(s):  
Wen Jun Liu ◽  
Hua Bo Chen ◽  
Alexander A. Khromykh
Keyword(s):  
Amino Acid ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

scholarly journals Mutational Analysis of the Tup1 General Repressor of Yeast

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 637-644
Author(s):  
Pauline M Carrico ◽  
Richard S Zitomer
Keyword(s):  
Mating Type ◽  
Transcriptional Repression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Repeat Region ◽  
Identify Amino Acid ◽  
Repression Complex ◽  
Mating Type Genes

Abstract The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.

scholarly journals Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

2001 ◽  
Vol 183 (19) ◽  
pp. 5743-5746 ◽  
Author(s):  
Babu S. Antharavally ◽  
Russell R. Poyner ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Paramagnetic Resonance ◽  
Specific Amino Acid ◽  
Dinitrogenase Reductase ◽  
Electron Paramagnetic ◽  
Hydrolysis Of

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

scholarly journals Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

1993 ◽  
Vol 177 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Y Lobet ◽  
C Feron ◽  
G Dequesne ◽  
E Simoen ◽  
P Hauser ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Pertussis Toxin ◽  
Neutralizing Antibodies ◽  
Whooping Cough ◽  
Site Directed Mutagenesis ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
Generation Vaccine ◽  
Adp Ribosyltransferase

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

scholarly journals Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

2005 ◽  
Vol 187 (5) ◽  
pp. 1552-1558 ◽  
Author(s):  
Masato Otsuka ◽  
Makoto Yasuda ◽  
Yuji Morita ◽  
Chie Otsuka ◽  
Tomofusa Tsuchiya ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ethidium Bromide ◽  
Vibrio Parahaemolyticus ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
Transmembrane Region ◽  
Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

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