scholarly journals Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli

1994 ◽  
Vol 298 (3) ◽  
pp. 675-680 ◽  
Author(s):  
J F Shaw ◽  
R C Chang ◽  
K H Chuang ◽  
Y T Yen ◽  
Y J Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cholesterol Acyltransferase ◽  
Reading Frame ◽  
Gene Coding ◽  
Step Procedure ◽  
Sds Page ◽  
Vibrio Mimicus ◽  
Hydrophobic Amino Acids ◽  
Cloned Gene

A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300. The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay. On the other hand, the activity was not lost when Ser-31 was changed to alanine. The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure. The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100. This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2). N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.

scholarly journals Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1454
Author(s):  
Yaretzi J. Pedroza-Gómez ◽  
Raquel Cossio-Bayugar ◽  
Hugo Aguilar-Díaz ◽  
Silvana Scarcella ◽  
Enrique Reynaud ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Dna Sequences ◽  
Bioinformatics Analysis ◽  
Fasciola Hepatica ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cellular Membrane ◽  
Reading Frame ◽  
Protein Mass ◽  
Gene Coding ◽  
Sds Page

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

scholarly journals Identification of the ubiD Gene on theEscherichia coli Chromosome

2000 ◽  
Vol 182 (21) ◽  
pp. 6243-6246 ◽  
Author(s):  
Haitao Zhang ◽  
George T. Javor
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gene Product ◽  
Mutant Strain ◽  
Amino Acid Residue ◽  
Open Reading Frame ◽  
Reading Frame

ABSTRACT The open reading frame at 86.7 min on the Escherichia coli chromosome, “yigC,” complemented aubiD mutant strain, AN66, indicating that yigCis the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

scholarly journals Cloning, Nucleotide Sequence, and Expression of theBrucella melitensis sucB Gene Coding for an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein

2001 ◽  
Vol 69 (10) ◽  
pp. 6537-6540 ◽  
Author(s):  
Michel S. Zygmunt ◽  
Marı́a A. Dı́az ◽  
Ana P. Teixeira-Gomes ◽  
Axel Cloeckaert
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Brucella Melitensis ◽  
Infected Sheep ◽  
Immunogenic Protein ◽  
Gene Encoding ◽  
Homologous Protein ◽  
Gene Coding ◽  
Dihydrolipoamide Succinyltransferase ◽  
Cloned Gene

ABSTRACT The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus andEscherichia coli, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Cloning of a Mucin-Desulfating Sulfatase Gene fromPrevotella Strain RS2 and Its Expression Using aBacteroides Recombinant System

2000 ◽  
Vol 182 (11) ◽  
pp. 3002-3007 ◽  
Author(s):  
Damian P. Wright ◽  
Catriona G. Knight ◽  
Shanthi G. Parkar ◽  
David L. Christie ◽  
Anthony M. Roberton
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Signal Sequence ◽  
Active Form ◽  
Open Reading Frame ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

ABSTRACT A gene encoding the mucin-desulfating sulfatase inPrevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into aBacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

scholarly journals Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli

1992 ◽  
Vol 288 (2) ◽  
pp. 649-655 ◽  
Author(s):  
N Ioannidis ◽  
C E Cooper ◽  
R K Poole
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Spectroscopic Studies ◽  
Oxygen Binding ◽  
Histidine Residue ◽  
Nitrosyl Complex ◽  
Sds Page ◽  
Prosthetic Groups

The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps. The prosthetic groups are identified as FAD and protohaem IX. SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene. The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm. The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively. Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm. This oxy form is not stable and slowly decays to the ferric state. Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r. characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue.

scholarly journals A Tetrahydrofolate-Dependent O-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (6) ◽  
pp. 2030-2037 ◽  
Author(s):  
Tomokuni Abe ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Double Mutant ◽  
Open Reading Frame ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Demethylase Activity ◽  
Specific Activities

ABSTRACT Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.

scholarly journals Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme

1991 ◽  
Vol 278 (3) ◽  
pp. 883-886 ◽  
Author(s):  
K Takazawa ◽  
J Perret ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Amino Acid ◽  
Kinase Activity ◽  
Cloning And Expression ◽  
Fusion Product ◽  
Molecular Evidence ◽  
Reading Frame ◽  
Acid Protein ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3′ end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.

scholarly journals Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase

1998 ◽  
Vol 331 (3) ◽  
pp. 953-958 ◽  
Author(s):  
Hiro-omi TAMURA ◽  
Yuki HARADA ◽  
Atsushi MIYAWAKI ◽  
Katsuhiko MIKOSHIBA ◽  
Michio MATSUI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nasal Cavity ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Reading Frame ◽  
Residue Polypeptide

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).

Sign in / Sign up

Export Citation Format

Share Document