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.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine

2013 ◽  
Vol 78 (3) ◽  
pp. 252-259 ◽  
Author(s):  
V. I. Timofeev ◽  
S. A. Kuznetsov ◽  
V. Kh. Akparov ◽  
G. G. Chestukhina ◽  
I. P. Kuranova
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Thermoactinomyces Vulgaris ◽  
Carboxypeptidase T

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.

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.

Crystal structure of the hinge domain of Smchd1 reveals its dimerization mode and nucleic acid–binding residues

2020 ◽  
Vol 13 (636) ◽  
pp. eaaz5599 ◽  
Author(s):  
Kelan Chen ◽  
Richard W. Birkinshaw ◽  
Alexandra D. Gurzau ◽  
Iromi Wanigasuriya ◽  
Ruoyun Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Muscular Dystrophy ◽  
Three Dimensional ◽  
Underlying Mechanism ◽  
Structural Features ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Dimensional Structure ◽  
Nucleic Acid Binding ◽  
X Ray Crystallography ◽  
Hinge Domain

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.

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