scholarly journals Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

2006 ◽  
Vol 72 (3) ◽  
pp. 1817-1824 ◽  
Author(s):  
Kou-San Ju ◽  
Rebecca E. Parales
Keyword(s):  
Hydrogen Bond ◽  
Substrate Specificity ◽  
Active Site ◽  
Product Formation ◽  
Oxidation Products ◽  
Site Directed Mutagenesis ◽  
Initial Reaction ◽  
Active Site Residues ◽  
Aromatic Rings ◽  
Α Subunit

ABSTRACT Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.

scholarly journals Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

2000 ◽  
Vol 182 (6) ◽  
pp. 1641-1649 ◽  
Author(s):  
Rebecca E. Parales ◽  
Kyoung Lee ◽  
Sol M. Resnick ◽  
Haiyan Jiang ◽  
Daniel J. Lessner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Product Formation ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Substitutions ◽  
Active Site Residues ◽  
Naphthalene Dioxygenase ◽  
Wide Range

ABSTRACT The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation ofcis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

scholarly journals Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255098
Author(s):  
Ekaterina Yu. Bezsudnova ◽  
Alena Yu. Nikolaeva ◽  
Alina K. Bakunova ◽  
Tatiana V. Rakitina ◽  
Dmitry A. Suplatov ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Active Site ◽  
Active Sites ◽  
Site Directed Mutagenesis ◽  
Primary Amines ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

scholarly journals Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

1999 ◽  
Vol 274 (4) ◽  
pp. 2344-2349 ◽  
Author(s):  
Shinya Oue ◽  
Akihiro Okamoto ◽  
Takato Yano ◽  
Hiroyuki Kagamiyama
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Cumulative Effects ◽  
Active Site Residues

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

2021 ◽  
Author(s):  
Ryuji Yamazawa ◽  
Ritsuko Kuwana ◽  
Kenji Takeuchi ◽  
Hiromu Takamatsu ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Modification ◽  
Cysteine Protease ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Protease Gene ◽  
Active Site Residues ◽  
Protein Determination ◽  
35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

scholarly journals Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues

Biochemistry ◽  
2019 ◽  
Vol 58 (21) ◽  
pp. 2534-2541
Author(s):  
Paul F. Fitzpatrick ◽  
Vi Dougherty ◽  
Bishnu Subedi ◽  
Jesus Quilantan ◽  
Cynthia S. Hinck ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Isotope Effects ◽  
Active Site Residues ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

scholarly journals Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds

Glycobiology ◽  
1998 ◽  
Vol 8 (10) ◽  
pp. 1021-1028 ◽  
Author(s):  
G. Garcia-Casado ◽  
C. Collada ◽  
I. Allona ◽  
R. Casado ◽  
L. F. Pacios ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Directed Mutagenesis ◽  
Active Site Residues

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.

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