Site-directed mutagenesis of the regulatory domain of escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals 1 1Edited by A. R. Fersht

2000 ◽  
Vol 299 (4) ◽  
pp. 979-991 ◽  
Author(s):  
Vicente Fresquet ◽  
Paz Mora ◽  
Lourdes Rochera ◽  
Santiago Ramón-Maiques ◽  
Vicente Rubio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Allosteric Regulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Regulatory Domain ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens

2013 ◽  
Vol 59 (6) ◽  
pp. 374-379 ◽  
Author(s):  
Haitian Fang ◽  
Huiyan Liu ◽  
Ning Chen ◽  
Chenglin Zhang ◽  
Xixian Xie ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bacillus Amyloliquefaciens ◽  
De Novo ◽  
Feedback Inhibition ◽  
Large Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Uridine Monophosphate

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP’s feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

scholarly journals Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

1992 ◽  
Vol 267 (32) ◽  
pp. 22830-22836 ◽  
Author(s):  
K Ostanin ◽  
E.H. Harms ◽  
P.E. Stevis ◽  
R Kuciel ◽  
M.M. Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Phosphatase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
Sign in / Sign up

Export Citation Format

Share Document