scholarly journals Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity

2002 ◽  
Vol 365 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Carlos NOVO ◽  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Alda CLEMENTE ◽  
Paul R. BROWN
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Data Bank ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Models

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys166 is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase—fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

RPS: Repeats in Protein Sequences

2011 ◽  
Vol 44 (3) ◽  
pp. 647-650
Author(s):  
Venkatesh Babu ◽  
M. Uthayakumar ◽  
M. Kirti Vaishnavi ◽  
R. Senthilkumar ◽  
M. Shankar ◽  
...  
Keyword(s):  
Structure Prediction ◽  
World Wide ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Data Bank ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Genome Database ◽  
Computing Resource ◽  
The World

Repeats are two or more contiguous segments of amino acid residues that are believed to have arisen as a result of intragenic duplication, recombination and mutation events. These repeats can be utilized for protein structure prediction and can provide insights into the protein evolution and phylogenetic relationship. Therefore, to aid structural biologists and phylogeneticists in their research, a computing resource (a web server and a database), Repeats in Protein Sequences (RPS), has been created. Using RPS, users can obtain useful information regarding identical, similar and distant repeats (of varying lengths) in protein sequences. In addition, users can check the frequency of occurrence of the repeats in sequence databases such as the Genome Database, PIR and SWISS-PROT and among the protein sequences available in the Protein Data Bank archive. Furthermore, users can view the three-dimensional structure of the repeats using the Java visualization plug-inJmol. The proposed computing resource can be accessed over the World Wide Web at http://bioserver1.physics.iisc.ernet.in/rps/.

scholarly journals PROCARB: A Database of Known and Modelled Carbohydrate-Binding Protein Structures with Sequence-Based Prediction Tools

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Adeel Malik ◽  
Ahmad Firoz ◽  
Vivekanand Jha ◽  
Shandar Ahmad
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Solvent Accessibility ◽  
Protein Structures ◽  
Three Dimensional ◽  
Data Bank ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Structural And Functional Properties ◽  
Three Dimensional Models

Understanding of the three-dimensional structures of proteins that interact with carbohydrates covalently (glycoproteins) as well as noncovalently (protein-carbohydrate complexes) is essential to many biological processes and plays a significant role in normal and disease-associated functions. It is important to have a central repository of knowledge available about these protein-carbohydrate complexes as well as preprocessed data of predicted structures. This can be significantly enhanced by tools de novo which can predict carbohydrate-binding sites for proteins in the absence of structure of experimentally known binding site. PROCARB is an open-access database comprising three independently working components, namely, (i) Core PROCARB module, consisting of three-dimensional structures of protein-carbohydrate complexes taken from Protein Data Bank (PDB), (ii) Homology Models module, consisting of manually developed three-dimensional models of N-linked and O-linked glycoproteins of unknown three-dimensional structure, and (iii) CBS-Pred prediction module, consisting of web servers to predict carbohydrate-binding sites using single sequence or server-generated PSSM. Several precomputed structural and functional properties of complexes are also included in the database for quick analysis. In particular, information about function, secondary structure, solvent accessibility, hydrogen bonds and literature reference, and so forth, is included. In addition, each protein in the database is mapped to Uniprot, Pfam, PDB, and so forth.

scholarly journals The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

2004 ◽  
Vol 380 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Young Sam SEO ◽  
Ahrim YOO ◽  
Jinwon JUNG ◽  
Soon-Kee SUNG ◽  
Dae Ryook YANG ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Comparative Modelling

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight α-helices, 12 β-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called ‘2-His-1-carboxylate facial triad’. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

scholarly journals Probing the Determinants of Coenzyme Specificity in Ferredoxin-NADP+Reductase by Site-directed Mutagenesis

2001 ◽  
Vol 276 (15) ◽  
pp. 11902-11912 ◽  
Author(s):  
Milagros Medina ◽  
Alejandra Luquita ◽  
Jesús Tejero ◽  
Juan Hermoso ◽  
Tomás Mayoral ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Structure Comparison ◽  
Coenzyme Specificity ◽  
Related Family ◽  
Secondary Interactions ◽  
Main Determinants

On the basis of sequence and three-dimensional structure comparison betweenAnabaenaPCC7119 ferredoxin-NADP+reductase (FNR) and other reductases from its structurally related family that bind either NADP+/H or NAD+/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2′-phosphate of NADP+/H inAnabaenaFNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP+/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions inAnabaenaFNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2′-phosphate NADP+/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261–268 inAnabaenaFNR must be one of these regions.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1042
Author(s):  
Jing-Jing Chen ◽  
Xiao Liang ◽  
Tian-Jiao Chen ◽  
Jin-Ling Yang ◽  
Ping Zhu
Keyword(s):  
Amino Acid ◽  
Lentinula Edodes ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Recent Success ◽  
Key Residues ◽  
Positive Effect ◽  
Candidate Strain

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

Critical roles of amino acids Ser231, His107 and Asp156 of Staphylococcus sciuri exfoliative toxin C (ExhC) in the induction of skin exfoliations in neonate mice

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Haihua Li ◽  
Fei He ◽  
Ziding Zhang ◽  
Shijun Zheng
Keyword(s):  
Active Site ◽  
Cultured Cells ◽  
Critical Role ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Newborn Mice ◽  
Exfoliative Toxin ◽  
Human Infections

AbstractStaphylococcus sciuri is a rare pathogen in humans, but it can cause a wide array of human infections. Recently a strain of S. sciuri (HBXX06) carrying exfoliative toxin C (ExhC) was reported to cause fatal exudative epidermitis in piglets and might be considered as a potential zoonotic agent. However, little is known regarding the pathogenicity of this bacterium. In this study, we predicted the three-dimensional structure of S. sciuri HBXX06 ExhC and replaced Ser231 or His107 or Asp156 in the active site of ExhC by site-directed mutagenesis, and examined the effects of mutant ExhC on BHK-21 cells and newborn mice as models. Interestingly, we found that mutant ExhC lost its exfoliative effects on newborn mice but could still induce necrosis in cultured cells if any one of the three amino acid residues in the active site was replaced. These results suggest that Ser231, His107 and Asp156 of ExhC play a critical role in the induction of skin exfoliation in neonate mice, which may help to further understand the mechanisms underlying the actions of exfoliative toxins.

scholarly journals Reaction Mechanism of Hydroxynitrile Lyases of the α/β-Hydrolase Superfamily

2004 ◽  
Vol 279 (19) ◽  
pp. 20501-20510 ◽  
Author(s):  
Karl Gruber ◽  
Günter Gartler ◽  
Barbara Krammer ◽  
Helmut Schwab ◽  
Christoph Kratky
Keyword(s):  
Reaction Mechanism ◽  
Active Site ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Acetone Cyanohydrin ◽  
Wild Type ◽  
Three Dimensional Structure ◽  
Catalytic Function

The hydroxynitrile lyases (HNLs) fromHevea brasiliensis(HbHNL) and fromManihot esculenta(MeHNL) are both members of the α/β-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for theHeveaenzyme but denied for theManihotenzyme. We applied a freeze-quench method to prepare crystals of the complex ofHbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys236the catalytic base His235would be protonated at neutral pH. All of this suggests that Lys236is instrumental for catalysis in several ways,i.e.by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN-, and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of α/β-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

scholarly journals Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1′ subsite from pancreatic carboxypeptidase B

2018 ◽  
Vol 74 (10) ◽  
pp. 638-643 ◽  
Author(s):  
Valery Kh. Akparov ◽  
Vladimir I. Timofeev ◽  
Inna P. Kuranova ◽  
Tatiana V. Rakitina
Keyword(s):  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Carboxypeptidase B ◽  
X Ray Crystallography ◽  
Thermoactinomyces Vulgaris ◽  
The Difference ◽  
A Site ◽  
Carboxypeptidase T

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1′ subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1′ subsite was crystallized and its three-dimensional structure was determined at 1.29 Å resolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1′ subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1′ subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1′ subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

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