Photoaffinity Labeling with the Activator IMP and Site-Directed Mutagenesis of Histidine 995 of Carbamoyl Phosphate Synthetase fromEscherichia coliDemonstrate That the Binding Site for IMP Overlaps with That for the Inhibitor UMP†

Biochemistry ◽  
1999 ◽  
Vol 38 (13) ◽  
pp. 3910-3917 ◽  
Author(s):  
Jorge Bueso ◽  
Javier Cervera ◽  
Vicente Fresquet ◽  
Alberto Marina ◽  
Carol J. Lusty ◽  
...  
Keyword(s):  
Binding Site ◽  
Photoaffinity Labeling ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
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 A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis

1991 ◽  
Vol 266 (24) ◽  
pp. 16105-16112
Author(s):  
M. Nikkola ◽  
F.K. Gleason ◽  
M. Saarinen ◽  
T. Joelson ◽  
O. Björnberg ◽  
...  
Keyword(s):  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Glutathione Binding
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