Characterization of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of detergent or phospholipid for optimal activity

Biochemistry ◽  
1995 ◽  
Vol 34 (12) ◽  
pp. 3935-3941 ◽  
Author(s):  
William Tschantz ◽  
Mark Paetzel ◽  
Guoqing Cao ◽  
Dominic Suciu ◽  
Masayori Inouye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Form ◽  
Optimal Activity ◽  
Catalytically Active ◽  
Leader Peptidase

scholarly journals Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase

1998 ◽  
Vol 334 (1) ◽  
pp. 219-224 ◽  
Author(s):  
James M. LAWTON ◽  
Shawn DOONAN
Keyword(s):  
Escherichia Coli ◽  
Aspartate Aminotransferase ◽  
Thermal Inactivation ◽  
Active Form ◽  
Active Conformation ◽  
E Coli ◽  
Catalytically Active ◽  
Mitochondrial Aspartate Aminotransferase ◽  
Chaperonin 10 ◽  
Chaperonin 60

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.

scholarly journals Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

2001 ◽  
Vol 67 (4) ◽  
pp. 1601-1606 ◽  
Author(s):  
Mitsunori Ishiguro ◽  
Satoshi Kaneko ◽  
Atsushi Kuno ◽  
Yoshinori Koyama ◽  
Shigeki Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Catalytic Function ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

Crystallization of a soluble, catalytically active form ofEscherichia coli leader peptidase

1995 ◽  
Vol 23 (1) ◽  
pp. 122-125 ◽  
Author(s):  
Mark Paetzel ◽  
Maia Chernaia ◽  
Natalie Strynadka ◽  
William Tschantz ◽  
Gnoqing Cao ◽  
...  
Keyword(s):  
Active Form ◽  
Ofescherichia Coli ◽  
Catalytically Active ◽  
Leader Peptidase

scholarly journals Characterization of the melA Locus for α-Galactosidase in Lactobacillus plantarum

2002 ◽  
Vol 68 (11) ◽  
pp. 5464-5471 ◽  
Author(s):  
Aurelio Silvestroni ◽  
Cristelle Connes ◽  
Fernando Sesma ◽  
Graciela Savoy de Giori ◽  
Jean-Christophe Piard
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Lactobacillus Plantarum ◽  
Posttranscriptional Regulation ◽  
Genetic Characterization ◽  
Active Form ◽  
Streptococcus Thermophilus ◽  
Transcriptional Level ◽  
High Homology

ABSTRACT Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of α-galactosidase (α-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous α-Gal is promising. In this framework, we characterized the melA gene for α-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active α-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both α- and β-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric β-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.

scholarly journals Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli

2007 ◽  
Vol 12 (5) ◽  
pp. 603-613 ◽  
Author(s):  
Wade C. McGregor ◽  
Sabina I. Swierczek ◽  
Brian Bennett ◽  
Richard C. Holz
Keyword(s):  
Escherichia Coli ◽  
Catalytically Active

scholarly journals In vitro characterization of the colibactin-activating peptidase ClbP enables development of a fluorogenic activity probe

2019 ◽  
Author(s):  
Matthew R. Volpe ◽  
Matthew R. Wilson ◽  
Carolyn A. Brotherton ◽  
Ethan S. Winter ◽  
Sheila E. Johnson ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inner Membrane ◽  
Substrate Preferences ◽  
In Vitro Characterization ◽  
Catalytically Active ◽  
Membrane Bound

ABSTRACTThe gut bacterial genotoxin colibactin has been linked to the development of colorectal cancer. In the final stages of colibactin’s biosynthesis, an inactive precursor (pre-colibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

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