Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.
Brizio
Carmen
C
Dipartimento di Biochimica e Biologia Molecolare Ernesto Quagliariello, Università degli Studi di Bari, Via Orabona 4, I-70126 Bari, Italy.
Galluccio
Michele
M
Wait
Robin
R
Torchetti
Enza Maria
EM
Bafunno
Valeria
V
Accardi
Rosita
R
Gianazza
Elisabetta
E
Indiveri
Cesare
C
Barile
Maria
M
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase