Over-expression in Escherichia coli, purification and characterization of isoform 2 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. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.
Galluccio
Michele
M
Dipartimento di Biologia Cellulare, Università della Calabria, Via P. Bucci 4c, I-87036 Arcavacata di Rende, Italy.
Brizio
Carmen
C
Torchetti
Enza Maria
EM
Ferranti
Pasquale
P
Gianazza
Elisabetta
E
Indiveri
Cesare
C
Barile
Maria
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000096:biosynthesis; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase