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+/-0.3muM. 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.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase / C. Brizio, M. Galluccio, R. Wait, E. M. Torchetti, V. Bafunno, R. Accardi, E. Gianazza, C. Indiveri, M. Barile. - In: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. - ISSN 0006-291X. - 344:3(2006), pp. 1008-1016.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

E. Gianazza;
2006

Abstract

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+/-0.3muM. 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.
Settore BIO/10 - Biochimica
2006
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/5379
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