Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli.
Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.
Olchowy
Jarosław
J
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, 11/12 Narutowicza St., 80-952 Gdańsk, Poland.
Kur
Krzysztof
K
Sachadyn
Paweł
P
Milewski
Sławomir
S
D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)