Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering

1987 ◽  
Vol 193 (4) ◽  
pp. 803-813 ◽  
Author(s):  
Alan J. Russell ◽  
Paul G. Thomas ◽  
Alan R. Fersht
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Electrostatic Effects ◽  
Active Site Cleft

scholarly journals Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
A.E. Naas ◽  
A.K. MacKenzie ◽  
B. Dalhus ◽  
V.G.H. Eijsink ◽  
P.B. Pope
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Structural Features ◽  
Structure And Function ◽  
Dimensional Structure ◽  
Active Site Cleft ◽  
Polysaccharide Utilization Locus ◽  
And Function ◽  
Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

scholarly journals The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities

2018 ◽  
Vol 399 (10) ◽  
pp. 1223-1235 ◽  
Author(s):  
Andreas Porodko ◽  
Ana Cirnski ◽  
Drazen Petrov ◽  
Teresa Raab ◽  
Melanie Paireder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Substrate Specificities ◽  
Active Site Cleft ◽  
Molecular Modeling Studies ◽  
Human Cathepsin

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Stefin A displaces the occluding loop of cathepsin B only by as much as required to bind to the active site cleft

FEBS Journal ◽  
2010 ◽  
Vol 277 (20) ◽  
pp. 4338-4345 ◽  
Author(s):  
Miha Renko ◽  
Urška Požgan ◽  
Dušana Majera ◽  
Dušan Turk
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Active Site Cleft ◽  
Stefin A

scholarly journals An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Swaminath Srinivas ◽  
John E. Cronan
Keyword(s):  
Fatty Acid ◽  
Structural Dynamics ◽  
Active Site ◽  
Lipid A ◽  
Acid Synthesis ◽  
Lipid Homeostasis ◽  
Temperature Sensitive ◽  
Active Site Cleft

ABSTRACT FabG performs the NADPH-dependent reduction of β-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (fabGΔ8) in Escherichia coli fabG that results from an in-frame 8-amino-acid residue deletion in the α6/α7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis both in vivo and in vitro. Surprisingly, the fabGΔ8 strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. The fabGΔ8 mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca2+ in vitro. We also observed a significant alteration in the lipid A fatty acid composition of fabGΔ8 strains but only in an lpxC background, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis. IMPORTANCE Membrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.

First-Sphere and Second-Sphere Electrostatic Effects in the Active Site of a Class Mu Glutathione Transferase†,‡

Biochemistry ◽  
1996 ◽  
Vol 35 (15) ◽  
pp. 4753-4765 ◽  
Author(s):  
Gaoyi Xiao ◽  
Suxing Liu ◽  
Xinhua Ji ◽  
William W. Johnson ◽  
Jihong Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Glutathione Transferase ◽  
Electrostatic Effects

Homology modeling and SAR analysis of Schistosoma japonicum cathepsin D (SjCD) with statin inhibitors identify a unique active site steric barrier with potential for the design of specific inhibitors

2005 ◽  
Vol 386 (4) ◽  
pp. 339-349 ◽  
Author(s):  
Conor R. Caffrey ◽  
Lenka Placha ◽  
Cyril Barinka ◽  
Martin Hradilek ◽  
Jiří Dostál ◽  
...  
Keyword(s):  
Schistosoma Japonicum ◽  
Active Site ◽  
Cathepsin D ◽  
Three Dimensional ◽  
Homology Model ◽  
Three Dimensional Model ◽  
Pathogenic Yeast ◽  
Active Site Cleft ◽  
Chronic Morbidity ◽  
Sar Analysis

Abstract Proteases that digest the blood-meal of the parasitic fluke Schistosoma are potential targets for therapy of schistosomiasis, a disease of chronic morbidity in humans. We generated a three-dimensional model of the cathepsin D target protease of Schistosoma japonicum (SjCD) utilizing the crystal structure of human cathepsin D (huCD) in complex with pepstatin as template. A homology model was also generated for the related secreted aspartic protease 2 (SAP2) of the pathogenic yeast, Candida albicans. An initial panel of seven statin inhibitors, originally designed for huCD [Majer et al., Protein Sci. 6 (1997), pp. 1458–1466], was tested against the two pathogen proteases. One inhibitor showed poor reactivity with SjCD. Examination of the SjCD active-site cleft revealed that the poor inhibition was due to a unique steric barrier situated between the S2 and S4 subsites. An in silico screen of 20 potential statin scaffolds with the SjCD model and incorporating the steric barrier constraint was performed. Four inhibitors (SJ1–SJ4) were eventually synthesized and tested with SjCD, bovine CD and SAP2. Of these, SJ2 and SJ3 proved moderately more specific for SjCD over bovine CD, with IC50 values of 15 and 60 nM, respectively. The unique steric barrier identified here provides a structural focus for further development of more specific SjCD inhibitors.

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