Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1988-11, Vol.263 (32), p.17050-17054
Main Authors: Eisermann, R, Deutscher, J, Gonzy-Treboul, G, Hengstenberg, W
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Bacillus subtilis
Bacillus subtilis - genetics
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Binding Sites
Biological and medical sciences
Biotechnology
Codon
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation
Genetic technics
Hot Temperature
Magnetic Resonance Spectroscopy
Methods. Procedures. Technologies
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Mutagenesis. Repair
Mutant screening
Phosphoenolpyruvate - metabolism
Phosphoenolpyruvate Sugar Phosphotransferase System - genetics
Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism
Phosphorylation
Prokaryotes
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)37496-9