Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

The CotA laccase fromBacillus subtilisis an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

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Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5290-5296.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5290-5296 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Yuan Liu ◽

Huabao Zheng ◽

Sheng Yang ◽

Weihong Jiang

Keyword(s):

Specific Activity ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Catalytic Function ◽

Alkaline Shift ◽

Key Residues

ABSTRACT In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k cat/Km ) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

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Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

Biochemical Journal ◽

10.1042/bj3490333 ◽

2000 ◽

Vol 349 (1) ◽

pp. 333-342 ◽

Cited By ~ 5

Author(s):

Gyles COZIER ◽

Richard SESSIONS ◽

Joanna R. BOTTOMLEY ◽

Jon S. REYNOLDS ◽

Peter J. CULLEN

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Inositol Phosphate ◽

Site Directed Mutagenesis ◽

Pleckstrin Homology Domain ◽

Directed Mutagenesis ◽

Ph Domain ◽

Pleckstrin Homology ◽

Gtpase Activating Protein ◽

Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

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Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli

Biochemical Journal ◽

10.1042/bj2530801 ◽

1988 ◽

Vol 253 (3) ◽

pp. 801-807 ◽

Cited By ~ 16

Author(s):

A M Gronenborn ◽

R Sandulache ◽

S Gärtner ◽

G M Clore

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Binding Site ◽

Cyclic Nucleotides ◽

Binding Pocket ◽

Receptor Protein ◽

Directed Mutagenesis ◽

Wild Type ◽

Cyclic Amp Receptor Protein ◽

Better Than

Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis. They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested. In addition, the binding of cyclic nucleotides to the mutants has been investigated. It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62→Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83→Ala, Ser-83→Lys, Thr-127→Ala, Ser-129→Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82→Leu). Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed.

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Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases

Biochemical Journal ◽

10.1042/bj3170141 ◽

1996 ◽

Vol 317 (1) ◽

pp. 141-145 ◽

Cited By ~ 22

Author(s):

Akiko HAMAHATA ◽

Yoshimi TAKATA ◽

Tomoharu GOMI ◽

Motoji FUJIOKA

Keyword(s):

Nucleic Acid ◽

Binding Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Sequence Motifs ◽

Cd Spectra ◽

Wild Type ◽

Wild Type Enzyme ◽

Aromatic Side Chain ◽

A Minor

Most mammalian non-nucleic acid methyltransferases share three sequence motifs. To gain insight into the S-adenosylmethionine (AdoMet)-binding site of guanidinoacetate methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly67 and Gly69) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr136 of motif II to a valine resulted in a decrease in kcat/Km values for substrates. Changing this residue to a phenylalanine caused only a minor change in kcat/Km for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu89, which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of guanidinoacetate methyltransferase.

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Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

Thrombosis and Haemostasis ◽

10.1160/th03-06-0392 ◽

2004 ◽

Vol 91 (01) ◽

pp. 111-118 ◽

Cited By ~ 4

Author(s):

Tatsushiro Tamura ◽

Jun Yamanouchi ◽

Shigeru Fujita ◽

Takaaki Hato

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Binding Site ◽

Thrombus Formation ◽

Direct Interaction ◽

Binding Pocket ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

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Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

International Journal of Molecular Sciences ◽

10.3390/ijms20246224 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6224 ◽

Cited By ~ 2

Author(s):

Bangqiao Yin ◽

Qinyan Hui ◽

Muhammad Kashif ◽

Ran Yu ◽

Si Chen ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Activity ◽

Directed Evolution ◽

Catalytic Efficiency ◽

Raw Material ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Substitutions ◽

The Novel ◽

Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

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Functional Analysis of DNA Gyrase Mutant Enzymes Carrying Mutations at Position 88 in the A Subunit Found in Clinical Strains of Mycobacterium tuberculosis Resistant to Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00944-06 ◽

2006 ◽

Vol 50 (12) ◽

pp. 4170-4173 ◽

Cited By ~ 35

Author(s):

Stéphanie Matrat ◽

Nicolas Veziris ◽

Claudine Mayer ◽

Vincent Jarlier ◽

Chantal Truffot-Pernot ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Dna Gyrase ◽

Multidrug Resistant ◽

Site Directed Mutagenesis ◽

Fluoroquinolone Resistance ◽

Directed Mutagenesis ◽

The Novel ◽

Wild Type ◽

Clinical Strains ◽

A Subunit

ABSTRACT We investigated the enzymatic efficiency and inhibition by quinolones of Mycobacterium tuberculosis DNA gyrases carrying the previously described GyrA G88C mutation and the novel GyrA G88A mutation harbored by two multidrug-resistant clinical strains and reproduced by site-directed mutagenesis. Fluoroquinolone MICs and 50% inhibitory concentrations for both mutants were 2- to 43-fold higher than for the wild type, demonstrating that these mutations confer fluoroquinolone resistance in M. tuberculosis.

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Resetting the ligand binding site of placental protein 13/galectin-13 recovers its ability to bind lactose

Bioscience Reports ◽

10.1042/bsr20181787 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 4

Author(s):

Jiyong Su ◽

Linlin Cui ◽

Yunlong Si ◽

Chenyang Song ◽

Yuying Li ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Crystal Structures ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ligand Binding Site ◽

Wild Type ◽

Placental Protein ◽

High Concentrations ◽

Lower Expression

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to β-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.

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Structural and biochemical analyses of theStreptococcus pneumoniaeL,D-carboxypeptidase DacB

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714025371 ◽

2015 ◽

Vol 71 (2) ◽

pp. 283-292

Author(s):

Juan Zhang ◽

Yi-Hu Yang ◽

Yong-Liang Jiang ◽

Cong-Zhao Zhou ◽

Yuxing Chen

Keyword(s):

Crystal Structure ◽

Molecular Docking ◽

Catalytic Mechanism ◽

Substrate Recognition ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Binding Properties ◽

Biochemical Analyses ◽

Key Residues ◽

Structural Insights

The L,D-carboxypeptidase DacB plays a key role in the remodelling ofStreptococcus pneumoniaepeptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB fromS. pneumoniaeTIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB fromS. pneumoniaeD39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, withKmandkcatvalues of 2.84 ± 0.37 mMand 91.49 ± 0.05 s−1, respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

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