scholarly journals Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1H-Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation

2010 ◽  
Vol 76 (9) ◽  
pp. 2940-2945 ◽  
Author(s):  
Gunjan Pandey ◽  
Susan J. Dorrian ◽  
Robyn J. Russell ◽  
Clint Brearley ◽  
Steven Kotsonis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Genetics ◽  
Biochemical Characterization ◽  
Hypothetical Protein ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Amino Acid Residues ◽  
Serine Hydrolase ◽  
Freeze Dried ◽  
Cognate Gene

ABSTRACT A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a Km and k cat of 6.1 μM and 170 min−1, respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

Characterization and Induction of Two Cytochrome P450 Genes, CYP6AE28 and CYP6AE30, in Cnaphalocrocis medinalis: Possible Involvement in Metabolism of Rice Allelochemicals

2010 ◽  
Vol 65 (11-12) ◽  
pp. 719-725 ◽  
Author(s):  
Xiaoli Liu ◽  
Jun Chen ◽  
Zhifan Yang
Keyword(s):  
Amino Acid ◽  
Rice Variety ◽  
Amino Acid Identity ◽  
Cnaphalocrocis Medinalis ◽  
Amino Acid Residues ◽  
Acid Identity ◽  
Rice Leaf Folder ◽  
Cytochrome P450 Genes ◽  
Molecular Masses ◽  
Lepidoptera Pyralidae

Two cDNAs specific for P450 genes, CYP6AE28 and CYP6AE30, have been isolated from the rice leaf folder Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae). Both cDNApredicted proteins have 504 amino acid residues in length, but with molecular masses of 60177 Dalton for CYP6AE28 and 60020 Dalton for CYP6AE30, and theoretical pI values of 8.49 for CYP6AE28 and 8.56 for CYP6AE30, respectively. Both putative proteins contain the conserved structural and functional domains characteristic of all CYP6 members. CYP6AE28 and CYP6AE30 show 52% amino acid identity to each other; both of them have 49 - 56% identities with CYP6AE1, Cyp6ae12, and CYP6AE14. Phylogenetic analysis showed that the two P450s are grouped in the lineage containing some of the CYP6AE members, CYP6B P450s and CYP321A1. The transcripts of CYP6AE28 and CYP6AE30 were found to be induced in response to TKM-6, a rice variety with high resistance to C. medinalis. The results suggest that the two P450s may play important roles in adaptation to the host plant rice. This is the first report of P450 genes cloned in C. medinalis

Comparative sequence analysis of morbillivirus receptors and its implication in host range expansion

2019 ◽  
Vol 65 (11) ◽  
pp. 783-794
Author(s):  
Ajay Kumar Yadav ◽  
Kaushal Kishor Rajak ◽  
Mukesh Bhatt ◽  
Ashok Kumar ◽  
Soumendu Chakravarti ◽  
...  
Keyword(s):  
Amino Acid ◽  
Host Range ◽  
Range Expansion ◽  
Small Ruminants ◽  
Amino Acid Identity ◽  
Amino Acid Residues ◽  
Binding Region ◽  
Acid Identity ◽  
Host Range Expansion ◽  
High Amino Acid

SLAM (CD150) and nectin-4 are the major morbillivirus receptors responsible for virus pathogenesis and host range expansion. Recently, morbillivirus infections have been reported in unnatural hosts, including endangered species, posing a threat to their conservation. To understand the host range expansion of morbilliviruses, we generated the full-length sequences of morbillivirus receptors (goat, sheep, and dog SLAM, and goat nectin-4) and tried to correlate their role in determining host tropism. A high level of amino acid identity was observed between the sequences of related species, and phylogenetic reconstruction showed that the receptor sequences of carnivores, marine mammals, and small ruminants grouped separately. Analysis of the ligand binding region (V region; amino acid residues 52–136) of SLAM revealed high amino acid identity between small ruminants and bovine SLAMs. Comparison of canine SLAM with ruminants and non-canids SLAM revealed appreciable changes, including charge alterations. Significant differences between feline SLAM and canine SLAM have been reported. The binding motifs of nectin-4 genes (FPAG motif and amino acid residues 60, 62, and 63) were found to be conserved in sheep, goat, and dog. The differences reported in the binding region may be responsible for the level of susceptibility or resistance of a species to a particular morbillivirus.

scholarly journals Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

2000 ◽  
Vol 182 (9) ◽  
pp. 2536-2543 ◽  
Author(s):  
Rachel A. L. Lassy ◽  
Charles G. Miller
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Serine Hydrolase ◽  
Genome Sequences ◽  
New Family ◽  
The Family ◽  
Serovar Typhimurium ◽  
Structural Classes

ABSTRACT Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available.

scholarly journals PC8 [corrected], a new member of the convertase family

1996 ◽  
Vol 314 (3) ◽  
pp. 727-731 ◽  
Author(s):  
Angela BRUZZANITI ◽  
Katrina GOODGE ◽  
Philippe JAY ◽  
Sylvie A. TAVIAUX ◽  
Mark H. C. Lam ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Duplication Event ◽  
Amino Acid Identity ◽  
Chromosome 15 ◽  
Rat Tissues ◽  
Amino Acid Residues ◽  
Degenerate Primers ◽  
Chromosome 11Q23 ◽  
Ancient Gene Duplication

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23–11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

scholarly journals Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages

2003 ◽  
Vol 77 (15) ◽  
pp. 8207-8215 ◽  
Author(s):  
Nathalie Vanderheijden ◽  
Peter L. Delputte ◽  
Herman W. Favoreel ◽  
Joël Vandekerckhove ◽  
Jozef Van Damme ◽  
...  
Keyword(s):  
Amino Acid ◽  
Viral Entry ◽  
Alveolar Macrophages ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Respiratory Syndrome Virus ◽  
Vesicle Membrane ◽  
Virus Particles ◽  
Internal Peptide ◽  
Peptide Data

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.

scholarly journals Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases

1999 ◽  
Vol 342 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Eiji ARIMITSU ◽  
Shinya AOKI ◽  
Syuhei ISHIKURA ◽  
Kumiko NAKANISHI ◽  
Kazuya MATSUURA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Japanese Monkey ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Animal Tissues ◽  
Dihydrodiol Dehydrogenase ◽  
Low Degrees

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Genetic and biochemical characterization of FRI-3, a novel variant of the Ambler class A carbapenemase FRI-1

2019 ◽  
Vol 74 (10) ◽  
pp. 2891-2894 ◽  
Author(s):  
Jennifer Schauer ◽  
Sören G Gatermann ◽  
Matthias Marschal ◽  
Niels Pfennigwerth
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Amino Acid Identity ◽  
Class A ◽  
Carbapenem Resistant ◽  
New Variant ◽  
Novel Variant ◽  
Almost All ◽  
Phenotypic Tests

Abstract Objectives To characterize a new variant of the FRI class A carbapenemase isolated from an MDR clinical Enterobacter cloacae isolate. Methods A carbapenem-resistant E. cloacae was isolated from a rectal swab from a patient in an ICU in Southern Germany. Various phenotypic tests confirmed production of a putative class A carbapenemase. The new bla gene variant, blaFRI-3, and its genetic environment were characterized by WGS. Biochemical characterization was performed by heterologous expression in Escherichia coli TOP10 and by purification of the enzyme with subsequent determination of its kinetic parameters. Results PCR and sequencing carried out for different class A carbapenemase genes confirmed the presence of a novel variant of blaFRI-1. The novel variant was named FRI-3 and exhibited 91%, 96% and 92% amino acid identity to FRI-1, FRI-2 and FRI-4, respectively. E. coli TOP10 expressing blaFRI-3 showed increased resistance to almost all β-lactams. Comparing the catalytic behaviour of FRI-3 and FRI-1, it was shown that FRI-3 had the same substrate spectrum, but basically hydrolysed β-lactams less efficiently than FRI-1. WGS data revealed that blaFRI-3 was located on a 111 kb plasmid. Conclusions The biochemical characterization of FRI-3 illustrates that even a few differences in the amino acid sequence can lead to altered catalytic activities of β-lactamases belonging to the same family.

scholarly journals Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

2016 ◽  
Vol 63 (1) ◽  
Author(s):  
Monika Wicka ◽  
Marta Wanarska ◽  
Ewelina Krajewska ◽  
Anna Pawlak-Szukalska ◽  
Józef Kur ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Pseudomonas Sp ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Optimal Activity ◽  
Cold Active ◽  
Psychrotolerant Bacterium ◽  
Type V

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

scholarly journals Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Sondes Mechri ◽  
Khelifa Bouacem ◽  
Meriam Amziane ◽  
Ahlem Dab ◽  
Farida Nateche ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Laundry Detergent ◽  
Biochemical Properties ◽  
White Shrimp ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Moderately Halophilic

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

scholarly journals cDNA cloning and amino acid sequence of human mitochondrial Δ3Δ2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase

1994 ◽  
Vol 300 (1) ◽  
pp. 1-5 ◽  
Author(s):  
J M Kilponen ◽  
H M Häyrinen ◽  
M Rehn ◽  
J K Hiltunen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basic Amino Acid ◽  
Peptide Sequencing ◽  
Amino Acid Residues ◽  
Mature Protein ◽  
Nucleotide Level ◽  
Sequence Identity ◽  
Human Enzyme

We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735. The human mitochondrial isomerase exhibits a 74% (78%) sequence identity with the corresponding rat counterpart at amino acid (nucleotide) level(s). Many basic amino acid residues in rat isomerase have been changed to acidic or neutral residues in human enzyme, explaining the differences observed between these proteins.

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