scholarly journals Carbamate C-N Hydrolase Gene ameH Responsible for the Detoxification Step of Methomyl Degradation in Aminobacter aminovorans Strain MDW-2

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Wankui Jiang ◽  
Chenfei Zhang ◽  
Qinqin Gao ◽  
Mingliang Zhang ◽  
Jiguo Qiu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Degradation Mechanism ◽  
Catalytic Efficiency ◽  
Acid Sites ◽  
Amino Acid Residues ◽  
Content Type ◽  
Key Enzyme ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Methomyl {bis[1-methylthioacetaldehyde-O-(N-methylcarbamoyl)oximino]sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain Aminobacter aminovorans MDW-2, and the encoding gene was termed ameH. A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from Schizosaccharomyces pombe ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. Km and kcat of AmeH for methomyl were 87.5 μM and 345.2 s−1, respectively, and catalytic efficiency (kcat/Km) was 3.9 μM−1 s−1. Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. IMPORTANCE Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and N-methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as cehA, mcbA, cahA, and mcd, and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, ameH, responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl.

scholarly journals Role of Asp104 in the SHV β-Lactamase

2006 ◽  
Vol 50 (12) ◽  
pp. 4124-4131 ◽  
Author(s):  
Christopher R. Bethel ◽  
Andrea M. Hujer ◽  
Kristine M. Hujer ◽  
Jodi M. Thomson ◽  
Mark W. Ruszczycky ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Amino Acid Residues ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Threefold Increase ◽  
Rate Limiting ◽  
Hydrolysis Of

ABSTRACT Among the TEM-type extended-spectrum β-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV β-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in β-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys β-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser β-lactamases. Correspondingly, the overall catalytic efficiency (k cat/Km ) of hydrolysis for cefotaxime was increased from 0.60 ± 0.07 μM−1 s−1 (mean ± standard deviation) for Gly238Ser to 1.70 ± 0.01 μM−1 s−1 for the Asp104Lys and Gly238Ser β-lactamase (threefold increase). We also showed that (i) k 3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser β-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 β-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

scholarly journals Peptidase Activity of Amidinated Thrombin

1977 ◽  
Author(s):  
E.P. Kang
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Human Thrombin ◽  
Small Change ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.

scholarly journals Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase

RSC Advances ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 5325-5337 ◽  
Author(s):  
Lubna Maryam ◽  
Shamsi Khalid ◽  
Abid Ali ◽  
Asad U. Khan
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Close Proximity

Mutations of amino acid residues present near active site decrease the catalytic efficiency of beta lactamase enzymes.

scholarly journals Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

2012 ◽  
Vol 78 (24) ◽  
pp. 8817-8821 ◽  
Author(s):  
Raushan Kumar Singh ◽  
Manish Kumar Tiwari ◽  
In-Won Kim ◽  
Zhilei Chen ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acid ◽  
Turnover Rate ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Wild Type ◽  
High Turnover ◽  
Substrate Binding Pocket ◽  
Content Type ◽  
Important Residue

ABSTRACTChaetomium globosumendo-1,4-β-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s−1), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg's catalytic efficiency.

scholarly journals Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

2005 ◽  
Vol 71 (7) ◽  
pp. 3420-3426 ◽  
Author(s):  
Helena Gradišar ◽  
Jožica Friedrich ◽  
Igor Križaj ◽  
Roman Jerala
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Paecilomyces Marquandii ◽  
Molecular Masses ◽  
Terminal Amino ◽  
Hydrolysis Of

ABSTRACT Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.

scholarly journals Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 β-lactamase hydrolysing third-generation cephalosporins

1998 ◽  
Vol 330 (3) ◽  
pp. 1443-1449 ◽  
Author(s):  
Anne-Typhaine BOUTHORS ◽  
Nathalie DAGONEAU-BLANCHARD ◽  
Thierry NAAS ◽  
Patrice NORDMANN ◽  
Vincent JARLIER ◽  
...  
Keyword(s):  
Marked Decrease ◽  
Residual Activity ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Important Residue ◽  
Bond Network ◽  
Hydrolysis Of ◽  
Third Generation Cephalosporins

The class A β-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum β-lactamases (ESBLs), catalyses the hydrolysis of oxyimino-β-lactams such as cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZT). Molecular modelling was used to identify in PER-1 the amino acid residues corresponding to those found at positions 104, 164, 238 and 240 in the TEM-type ESBLs, which are critical for hydrolysis of oxyimino-β-lactams. The function of these residues in PER-1 was assessed by site-directed mutagenesis. In this enzyme, residue 104 could be either a glutamine, an asparagine or a threonine. The Gln → Gly mutation did not significantly affect the catalytic efficiency, while Asn → Gly and Thr → Glu resulted in a marked decrease in catalytic activity, probably due to the alteration of a hydrogen bond network connecting the putative Asn-104 residue to Asn-132 and Glu-166. Replacement of Ala-164 by Arg in PER-1 resulted in a mutant with no detectable activity, thus suggesting that Ala-164 is important for catalysis and stability of PER-1. Conversely, Ser-238 → Gly and Gly-240 → Glu had little effect on kcat and Km values. Finally, the replacement of the catalytic residue Glu-166 by an alanine resulted in a complete loss of activity for CTX and a marked decrease of kcat for CAZ and AZT. These results suggest that Glu-166 is an important residue in PER-1. However, residues other than Glu-166 could contribute in maintaining residual activity towards oxyimino-β-lactams in the Ala-166 mutant.

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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