Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme

Biochemistry ◽  
1991 ◽  
Vol 30 (46) ◽  
pp. 11073-11080 ◽  
Author(s):  
Jeffrey O. Boles ◽  
Ralph J. Cisneros ◽  
Mary S. Weir ◽  
Jerome D. Odom ◽  
J. E. Villafranca ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thymidylate Synthase ◽  
Wild Type ◽  
Purification And Characterization ◽  
Wild Type Enzyme

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Purification and characterization of wild-type and ts112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli

Virology ◽  
1990 ◽  
Vol 175 (1) ◽  
pp. 222-231 ◽  
Author(s):  
M. Cuillel ◽  
B. Cortolezzis ◽  
J. Chroboczek ◽  
J. Langowski ◽  
R.W.H. Ruigrok ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Adenovirus ◽  
Mutant Protein ◽  
Wild Type ◽  
Purification And Characterization

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.

Production in Escherichia coli, folding, purification and characterization of notexin with wild type sequence and with N-terminal and catalytic site mutations

Toxicon ◽  
2014 ◽  
Vol 88 ◽  
pp. 11-20 ◽  
Author(s):  
Morena Simonato ◽  
Laura Morbiato ◽  
Veronica Zorzi ◽  
Paola Caccin ◽  
Julián Fernández ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Site ◽  
Wild Type ◽  
Purification And Characterization ◽  
Wild Type Sequence ◽  
Type Sequence

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

1995 ◽  
Vol 8 (3) ◽  
pp. 261-273 ◽  
Author(s):  
Stephen B. delCardayré ◽  
Marc Ribó ◽  
Erich M. Yokel ◽  
David J. Quirk ◽  
William J. Rutter ◽  
...  
Keyword(s):  
Ribonuclease A ◽  
Wild Type ◽  
Purification And Characterization ◽  
Wild Type Enzyme

scholarly journals Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1208 ◽  
Author(s):  
Mohamed H. Habib ◽  
Henriëtte J. Rozeboom ◽  
Marco W. Fraaije
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sequence Motif ◽  
Acidic Ph ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

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