Int. J. Mol. Sci. 2012, 13(12), 16880-16898; doi:10.3390/ijms131216880
Article

Bacterial Over-Expression and Purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

Received: 10 October 2012; in revised form: 14 November 2012 / Accepted: 20 November 2012 / Published: 11 December 2012
(This article belongs to the Special Issue Flavins)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ1-328-hFADS enzyme shows a slight, but not significant, change of Km values (0.24 and 6.23 µM for FMN and ATP, respectively) and of kcat (4.2 × 10−2 s−1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ1-328-hFADS versus wild-type protein.
Keywords: FLAD1; human FAD synthase; FMN adenylyltransferase; Flavin; FAD; PAPS reductase domain; molybdopterin-binding domain
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MDPI and ACS Style

Miccolis, A.; Galluccio, M.; Giancaspero, T.A.; Indiveri, C.; Barile, M. Bacterial Over-Expression and Purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling. Int. J. Mol. Sci. 2012, 13, 16880-16898.

AMA Style

Miccolis A, Galluccio M, Giancaspero TA, Indiveri C, Barile M. Bacterial Over-Expression and Purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling. International Journal of Molecular Sciences. 2012; 13(12):16880-16898.

Chicago/Turabian Style

Miccolis, Angelica; Galluccio, Michele; Giancaspero, Teresa A.; Indiveri, Cesare; Barile, Maria. 2012. "Bacterial Over-Expression and Purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling." Int. J. Mol. Sci. 13, no. 12: 16880-16898.

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