scholarly journals Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.

1994 ◽  
Vol 269 (10) ◽  
pp. 7450-7457
Author(s):  
S.K. Boehlein ◽  
N.G. Richards ◽  
S.M. Schuster
Keyword(s):  
Escherichia Coli ◽  
Catalytic Triad ◽  
Asparagine Synthetase ◽  
Nitrogen Transfer

scholarly journals An Efficient System for Intestinal On-site Butyrate Production Using Novel Microbiome-Derived Esterases

2021 ◽  
Author(s):  
Dah Hyun Jung ◽  
Ji Hyun Yong ◽  
Wontae Hwang ◽  
Mi Young Yoon ◽  
Sang Sun Yoon
Keyword(s):  
Escherichia Coli ◽  
Artificial Chromosome ◽  
Short Chain Fatty Acids ◽  
Catalytic Triad ◽  
Nucleotide Sequence Analysis ◽  
Efficient System ◽  
Gastrointestinal Health ◽  
Metagenome Library ◽  
The Family ◽  
Butyrate Production

Abstract Short-chain fatty acids, especially butyrate, play beneficial roles in sustaining gastrointestinal health. However, due to limitations associated with direct consumption of butyrate, there has been interest in using prodrugs of butyrate. Tributyrin (TB), a triglyceride composed of three butyrate molecules and a glycerol, is a well-studied precursor of butyrate. We screened a metagenome library consisting of 5,760 bacterial artificial chromosome clones, with DNA inserts originating from mouse microbiomes, and identified two clones that efficiently hydrolyse TB into butyrate. Nucleotide sequence analysis indicated that inserts in these two clones are derived from unknown microbes. BLASTp analysis, however, revealed that each insert contains a gene homologous to acetylesterase or esterase genes, from Clostridium spp. and Bacteroides spp., respectively. Predicted structures of these two proteins both contain serine-histidine-aspartate catalytic triad, highly conserved in the family of esterases. Escherichia coli host expressing each of the two candidate genes invariably produced greater amounts of butyrate in the presence of TB. Importantly, administration of TB together with cloned Escherichia coli cells alleviated inflammatory symptoms in a mouse model of acute colitis. Based on these results, we established an efficient on-site and real-time butyrate production system that releases butyrate in a controlled manner inside the intestine

Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells

1987 ◽  
Vol 7 (5) ◽  
pp. 1623-1628
Author(s):  
M Cartier ◽  
M W Chang ◽  
C P Stanners
Keyword(s):  
Escherichia Coli ◽  
Cell Lines ◽  
Animal Cell ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Asparagine Synthetase ◽  
Translational Initiation ◽  
Shuttle Vectors ◽  
Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

scholarly journals Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.

1987 ◽  
Vol 7 (5) ◽  
pp. 1623-1628 ◽  
Author(s):  
M Cartier ◽  
M W Chang ◽  
C P Stanners
Keyword(s):  
Escherichia Coli ◽  
Cell Lines ◽  
Animal Cell ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Asparagine Synthetase ◽  
Translational Initiation ◽  
Shuttle Vectors ◽  
Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

ChemInform Abstract: A Potent Transition-State Analogue Inhibitor of Escherichia coli Asparagine Synthetase A.

ChemInform ◽  
2010 ◽  
Vol 30 (39) ◽  
pp. no-no
Author(s):  
Mitsuteru Koizumi ◽  
Jun Hiratake ◽  
Toru Nakatsu ◽  
Hiroaki Kato ◽  
Jun'ichi Oda
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Asparagine Synthetase ◽  
Transition State Analogue

scholarly journals Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1

2006 ◽  
Vol 397 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Li-Chiun Lee ◽  
Ya-Lin Lee ◽  
Ruey-Jyh Leu ◽  
Jei-Fu Shaw
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Water Molecule ◽  
Acyl Chain ◽  
Residual Activity ◽  
Catalytic Triad ◽  
The Other ◽  
Oxyanion Hole

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.

scholarly journals NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

FEBS Letters ◽  
2002 ◽  
Vol 528 (1-3) ◽  
pp. 203-206 ◽  
Author(s):  
Sergiy I Tyukhtenko ◽  
Alexandra V Litvinchuk ◽  
Chi-Fon Chang ◽  
Ruey-Jyh Leu ◽  
Jei-Fu Shaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Bonds ◽  
Catalytic Triad ◽  
Nmr Studies

scholarly journals Functional Characterization of Escherichia coli GlpG and Additional Rhomboid Proteins Using an aarA Mutant of Providencia stuartii

2006 ◽  
Vol 188 (9) ◽  
pp. 3415-3419 ◽  
Author(s):  
Katy M. Clemmer ◽  
Gwen M. Sturgill ◽  
Alexander Veenstra ◽  
Philip N. Rather
Keyword(s):  
Escherichia Coli ◽  
Serine Proteases ◽  
Functional Characterization ◽  
Catalytic Triad ◽  
Cell Physiology ◽  
Phenotype Microarray ◽  
Cellular Functions ◽  
Extracellular Signaling ◽  
Lactam Antibiotic ◽  
Providencia Stuartii

ABSTRACT The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the β-lactam antibiotic cefotaxime.

Sign in / Sign up

Export Citation Format

Share Document