Characterization of Escherichia coli thioredoxins with altered active site residues

Biochemistry ◽  
1990 ◽  
Vol 29 (15) ◽  
pp. 3701-3709 ◽  
Author(s):  
Florence K. Gleason ◽  
Chang Jin Lim ◽  
Maryam Gerami-Nejad ◽  
James A. Fuchs
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Site Residues

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

2001 ◽  
Vol 357 (2) ◽  
pp. 399 ◽  
Author(s):  
Danuta MAKSEL ◽  
Paul R. GOOLEY ◽  
James D. SWARBRICK ◽  
Andrzej GURANOWSKI ◽  
Christine GANGE ◽  
...  
Keyword(s):  
Active Site ◽  
Lupinus Angustifolius ◽  
Active Site Residues ◽  
Diadenosine Tetraphosphate

scholarly journals Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Shangen Xu ◽  
Junwei Zhou ◽  
Yingjin Chen ◽  
Xue Tong ◽  
Zixin Wang ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Hydrophobic Force ◽  
Substrate Specificities ◽  
Consensus Sequences ◽  
Cleavage Activity ◽  
Porcine Torovirus

ABSTRACT The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase’s substrate specificities and the rational development of the antinidovirus drugs. IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The “noncanonical” substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

2006 ◽  
Vol 34 (1) ◽  
pp. 143-145 ◽  
Author(s):  
T.A. Clarke ◽  
A.M. Hemmings ◽  
B. Burlat ◽  
J.N. Butt ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Desulfovibrio Desulfuricans ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Wolinella Succinogenes ◽  
Nitrite Reductases ◽  
Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

Characterization of xenobiotic reductase A (XenA): study of active site residues, substrate spectrum and stability

2010 ◽  
Vol 46 (46) ◽  
pp. 8809 ◽  
Author(s):  
Yanto Yanto ◽  
Hua-Hsiang Yu ◽  
Mélanie Hall ◽  
Andreas S. Bommarius
Keyword(s):  
Active Site ◽  
Active Site Residues ◽  
Substrate Spectrum

scholarly journals Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

2012 ◽  
Vol 287 (15) ◽  
pp. 11788-11797 ◽  
Author(s):  
Huanchen Wang ◽  
Stefan Kunz ◽  
Gong Chen ◽  
Thomas Seebeck ◽  
Yiqian Wan ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Trypanosoma Evansi ◽  
Active Site Residues ◽  
Dual Specificity ◽  
Pde Inhibitors ◽  
Starting Model

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

scholarly journals Characterization of active site residues of nitroalkane oxidase

2010 ◽  
Vol 38 (3) ◽  
pp. 115-119 ◽  
Author(s):  
Michael P. Valley ◽  
Nana S. Fenny ◽  
Shah R. Ali ◽  
Paul F. Fitzpatrick
Keyword(s):  
Active Site ◽  
Active Site Residues

scholarly journals Enantioseparation, in vitro testing, and structural characterization of triple-binding reactivators of organophosphate-inhibited cholinesterases

2020 ◽  
Vol 477 (15) ◽  
pp. 2771-2790 ◽  
Author(s):  
Nikola Maraković ◽  
Anamarija Knežević ◽  
Igor Rončević ◽  
Xavier Brazzolotto ◽  
Zrinka Kovarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Structural Characterization ◽  
Active Site ◽  
In Vitro Testing ◽  
Active Site Residues

The enantiomers of racemic 2-hydroxyimino-N-(azidophenylpropyl)acetamide-derived triple-binding oxime reactivators were separated, and tested for inhibition and reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited with tabun (GA), cyclosarin (GF), sarin (GB), and VX. Both enzymes showed the greatest affinity toward the methylimidazole derivative (III) of 2-hydroxyimino-N-(azidophenylpropyl)acetamide (I). The crystal structure was determined for the complex of oxime III within human BChE, confirming that all three binding groups interacted with active site residues. In the case of BChE inhibited by GF, oximes I (kr = 207 M−1 min−1) and III (kr = 213 M−1 min−1) showed better reactivation efficiency than the reference oxime 2-PAM. Finally, the key mechanistic steps in the reactivation of GF-inhibited BChE with oxime III were modeled using the PM7R6 method, stressing the importance of proton transfer from Nε of His438 to Oγ of Ser203 for achieving successful reactivation.

scholarly journals FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

2019 ◽  
Author(s):  
Yashwanth Ashok ◽  
Mirko M. Maksimainen ◽  
Tuija Kallio ◽  
Pekka Kilpeläinen ◽  
Lari Lehtiö
Keyword(s):  
Hydrogen Peroxide ◽  
Active Site ◽  
Pediococcus Acidilactici ◽  
Active Site Residues ◽  
Lactate Oxidase ◽  
Monooxygenase Activity ◽  
Aerococcus Viridans ◽  
Lactate Levels

AbstractLactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

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