scholarly journals Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli : Characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase

1996 ◽  
Vol 5 (4) ◽  
pp. 709-718 ◽  
Author(s):  
Lauren B. Murata ◽  
H.K. Schachman
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Structural Similarity ◽  
Ornithine Transcarbamoylase ◽  
Carboxy Terminal

scholarly journals Characterization of Borrelia burgdorferiBlyA and BlyB Proteins: a Prophage-Encoded Holin-Like System

2000 ◽  
Vol 182 (23) ◽  
pp. 6791-6797 ◽  
Author(s):  
Christopher J. Damman ◽  
Christian H. Eggers ◽  
D. Scott Samuels ◽  
Donald B. Oliver
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Activity ◽  
Soluble Protein ◽  
Structural Similarity ◽  
Structural Features ◽  
Accessory Protein ◽  
E Coli ◽  
Bacteriophage Particle ◽  
Membrane Interactive

ABSTRACT The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308–7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. AnEscherichia coli mutant containing the blyABlocus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction ofsheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage inB. burgdorferi dramatically stimulated blyABexpression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

2006 ◽  
Vol 34 (1) ◽  
pp. 143-145 ◽  
Author(s):  
T.A. Clarke ◽  
A.M. Hemmings ◽  
B. Burlat ◽  
J.N. Butt ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Desulfovibrio Desulfuricans ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Wolinella Succinogenes ◽  
Nitrite Reductases ◽  
Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

Characterization of Escherichia coli thioredoxins with altered active site residues

Biochemistry ◽  
1990 ◽  
Vol 29 (15) ◽  
pp. 3701-3709 ◽  
Author(s):  
Florence K. Gleason ◽  
Chang Jin Lim ◽  
Maryam Gerami-Nejad ◽  
James A. Fuchs
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Site Residues

scholarly journals Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011 ◽  
Vol 193 (19) ◽  
pp. 5400-5411 ◽  
Author(s):  
L. A. Hawver ◽  
C. A. Gillooly ◽  
P. J. Beuning
Keyword(s):  
Escherichia Coli ◽  
Active Site

scholarly journals Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

1990 ◽  
Vol 271 (2) ◽  
pp. 487-491 ◽  
Author(s):  
A Hädener ◽  
P R Alefounder ◽  
G J Hart ◽  
C Abell ◽  
A R Battersby
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Kinetic Studies ◽  
Enzyme Kinetic ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Over Expression ◽  
Putative Active Site ◽  
Lysine Residues

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

scholarly journals Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (2) ◽  
pp. 375-381 ◽  
Author(s):  
G. Potocki De Montalk ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
V. Planchot ◽  
P. Monsan
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Active Site ◽  
Degree Of Polymerization ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
Strong Homology ◽  
Branched Chains ◽  
Sepharose 4B

ABSTRACT The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathioneS-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.

scholarly journals Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

2019 ◽  
Vol 20 (14) ◽  
pp. 3530 ◽  
Author(s):  
Ophelia Gevaert ◽  
Stevie Van Overtveldt ◽  
Koen Beerens ◽  
Tom Desmet
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Chemical Reactions ◽  
Active Site ◽  
Expression In Escherichia Coli ◽  
Thermophilic Organism ◽  
Sequence Databases

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.

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