scholarly journals Amino acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

2004 ◽  
Vol 383 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Sun-Mi JUNG ◽  
Dung Tien LE ◽  
Sung-Sook YOON ◽  
Moon-Young YOON ◽  
Young Tae KIM ◽  
...  
Keyword(s):  
Binding Site ◽  
Tertiary Structure ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Cross Resistance ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Acetohydroxy Acid Synthase ◽  
Herbicide Binding ◽  
Moderately Resistant

The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered Vmax values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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 Revealing two important tryptophan residues with completely different roles in a dye-decolorizing peroxidase from Irpex lacteus F17

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Liuqing Li ◽  
Tao Wang ◽  
Taohua Chen ◽  
Wenhan Huang ◽  
Yinliang Zhang ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
High Spin State ◽  
Site Directed Mutagenesis ◽  
Molecular Surface ◽  
Synthetic Dyes ◽  
Amino Acid Residues ◽  
Irpex Lacteus ◽  
Paramagnetic Resonance ◽  
High Resolution Structure ◽  
Heme Peroxidases

Abstract Background Dye-decolorizing peroxidases (DyPs) represent a novel family of heme peroxidases that use H2O2 as the final electron acceptor to catalyze the oxidation of various organic compounds. A DyP from Irpex lacteus F17 (Il-DyP4, corresponding to GenBank MG209114), obtained by heterologous expression, exhibits a high catalytic efficiency for phenolic compounds and a strong decolorizing ability toward various synthetic dyes. However, the enzyme structure and the catalytic residues involved in substrate oxidation remain poorly understood. Results Here, we obtained a high-resolution structure (2.0 Å, PDB: 7D8M) of Il‑DyP4 with α-helices, anti-parallel β-sheets and one ferric heme cofactor sandwiched between two domains. The crystal structure of Il‑DyP4 revealed two heme access channels leading from the enzyme molecular surface to its heme region, and also showed four conserved amino acid residues forming the pocket for the conversion of hydrogen peroxide into the water molecule. In addition, we found that Trp264 and Trp380, were two important residues with different roles in Il‑DyP4, by using site-directed mutagenesis and an electron paramagnetic resonance (EPR) study. Trp264 is a noncatalytic residue that mainly is used for maintaining the normal spatial conformation of the heme region and the high-spin state of heme Fe3+ of Il‑DyP4, while Trp380 serves as the surface-exposed radical-forming residue that is closely related to the oxidation of substrates including not only bulky dyes, but also simple phenols. Conclusions This study is important for better understanding the catalytic properties of fungal DyPs and their structure–function relationships.

scholarly journals Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

2000 ◽  
Vol 352 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Claire LEADBEATER ◽  
Lisa MCIVER ◽  
Dominic J. CAMPOPIANO ◽  
Scott P. WEBSTER ◽  
Robert L. BAXTER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Binding Site ◽  
Tertiary Structure ◽  
Reductase Activity ◽  
Close Approach ◽  
Pyridine Nucleotide ◽  
Phosphate Group ◽  
Site Directed Mutagenesis ◽  
Wild Type

The structure of the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6min-1; R174A, 132.1min-1; R184A, 305.5min-1 versus wild type, 338.9min-1) and increased Km for NADPH (R144A, 5.3µM; R174A, 20.2µM; R184A, 54.4µM versus wild type, 3.9µM). The kcat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3min-1) and R184A (50.4min-1) compared with the wild type (33.0min-1), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2′-phosphate. Stopped-flow studies indicated that affinity (Kd) for NADPH was markedly reduced in mutants R144A (635µM) and R184A (2.3mM) compared with the wild type (< 5µM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH Kd ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (kred = 8.82s-1 versus 22.63s-1 for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2′-phosphate group, and R144 likely to interact with the nicotinamide ribose 5′-phosphate group.

Catalytic activity of mutants of yeast protein kinase CK2alpha.

2008 ◽  
Vol 55 (4) ◽  
pp. 767-776 ◽  
Author(s):  
Ewa Sajnaga ◽  
Konrad Kubiński ◽  
Ryszard Szyszka
Keyword(s):  
Protein Kinase ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Kinetic Properties ◽  
Helical Structures ◽  
Hydrophobic Pocket

Yeast CK2 is a highly conserved member of the protein kinase CGMC subfamily composed of two catalytic (alpha and alpha') and two regulatory (beta and beta') subunits. The amino-acid sequences of both catalytic subunits are only 60% homologous. Modelling of the tertiary structure of the CK2alpha displays additional alpha-helical structures not present in the CK2alpha' subunit, connecting the ATP-binding loop with the catalytic and activation loops. Deletion of this part causes drastic structural and enzymatic changes of the protein (CK2alpha(Delta91-128)) with characteristics similar to yeast CK2alpha' (low sensitivity to salt, heparin and spermine). Additionally, the deletion causes an over 5-fold decrease of the binding affinity for ATP and ATP-competitive inhibitors (TBBt and TBBz). The structural basis for TBBt and TBBz selectivity is provided by the hydrophobic pocket adjacent to the ATP/GTP binding site, which is smaller in CK2 than in the majority of other protein kinases. The importance of hydrophobic interactions in the binding of specific inhibitors was investigated here by mutational analysis of CK2alpha residues whose side chains contribute to reducing the size of the hydrophobic pocket. Site-directed mutagenesis was used to replace Val67 and Ile213 by Ala. The kinetic properties of the single mutants CK2alpha(Val67Ala) and CK2alpha(Ile213Ala), and the double mutant CK2(Val67Ala Ile213Ala) were studied with respect to ATP, and both inhibitors TBBt and TBBz. The K(m) values for ATP did not change or were very close to those of the parental kinase. In contrast, all CK2alpha mutants analysed displayed higher K(i) values towards the inhibitors (10 to 12-fold higher with TBBt and 3 to 6-fold with TBBt) comparing to recombinant wild-type CK2alpha.

scholarly journals Structure-Based Engineering of Methionine Residues in the Catalytic Cores of Alkaline Amylase from Alkalimonas amylolytica for Improved Oxidative Stability

2012 ◽  
Vol 78 (21) ◽  
pp. 7519-7526 ◽  
Author(s):  
Haiquan Yang ◽  
Long Liu ◽  
Mingxing Wang ◽  
Jianghua Li ◽  
Nam Sun Wang ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Oxidative Stability ◽  
Tertiary Structure ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Original Activity ◽  
Alkaline Stability ◽  
Alkaline Amylase

ABSTRACTThis work aims to improve the oxidative stability of alkaline amylase fromAlkalimonas amylolyticathrough structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H2O2at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, thekcatvalues of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 104to 5.6 × 104min−1. The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

scholarly journals Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase

2017 ◽  
Vol 114 (47) ◽  
pp. 12530-12535 ◽  
Author(s):  
Victoria J. Laye ◽  
Ram Karan ◽  
Jong-Myoung Kim ◽  
Wolf T. Pecher ◽  
Priya DasSarma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Comparative Genomics ◽  
Active Site ◽  
Interdisciplinary Approach ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Temperature Dependent ◽  
Temperature Function ◽  
Cold Temperatures

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

scholarly journals Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

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