Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits.
Acetohydroxyacid synthase I (AHAS I), one of three isozymes in Escherichia coli catalyzing the first common step in the biosynthesis of branched amino acids, is composed of two kinds of subunits. The large catalytic (B) and small regulatory (N) subunits of the holoenzyme dissociate and associate freely and rapidly and are quite different in size, charge and hydrophobicity, so that high resolution purification methods lead to partial separation of subunits and to heterogeneity. We have prepared several linked AHAS I proteins, in which the large subunit B with a hexahistidine-tag at the N-terminus, was covalently joined by a flexible linker, containing several (X) amino acids, to the small subunit N to form His6-BuXN polypeptides. All linked BuXN polypeptides have similar specific activity, sensitivity to valine and substrate specificity as the wild type holoenzyme. The most successful BuXN linked protein (Bu30N-r) was inserted into and expressed in yeast and its catalytic properties were tested.
Vyazmensky
Maria
M
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. vyas@bgumail.bgu.ac.il
Engel
Stainslav
S
Kryukov
Olga
O
Berkovich-Berger
Dvora
D
Kaplun
Ludmila
L
D000095:Acetolactate Synthase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D000095:Acetolactate Synthase