Premium
Cloning of the phosphotransacetylase gene from Lactobacillus sanfranciscensis and characterization of its gene product
Author(s) -
Knorr Ruth,
Ehrmann Matthias A.,
Vogel Rudi F.
Publication year - 2001
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/1521-4028(200112)41:6<339::aid-jobm339>3.0.co;2-0
Subject(s) - cloning (programming) , gene , gene product , biology , chemistry , biochemistry , gene expression , computer science , programming language
The phosphotransacetylase (PTA) (EC 2.3.1.8) catalyzes a key branch point reaction in the carbohydrate pathway of Lactobacillus sanfranciscensis . In this report, we describe the cloning of the pta gene. The DNA sequence analysis revealed a 987 bp open reading frame encoding a protein with a molecular mass of 35.5 kD. These are the first studies on a PTA of an organism representative for the heterofermentative lactic acid bacteria. Unlike in most other bacteria analysed so far, in L. sanfranciscensis the pta gene is not adjacent located to the gene encoding acetatekinase. The PTA was heterologously expressed as a biotinylated fusion protein in E. coli and purified to homogeneity. Rate dependence on all substrates followed Michaelis‐Menten kinetics. The apparent K m values for acetylphosphate and CoA (forward reaction) were 1.3 and 0.1 m M , respectively. The apparent V max was 194 U/mg. The enzyme also catalyzed in vitro the reverse reaction with apparent K m values for acetylCoA and phosphate of 0.6 and 6.7 m M , respectively ( V max of 38 U/mg). The PTA showed a wide range of temperature for optimal activity (49 °C to 58 °C). It was inactivated after 15 min at 60 °C. Its activity was not affected by addition of MgCl 2 (10 m M ) or KCl (100 m M ).