Next Article in Journal / Special Issue
Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB
Previous Article in Journal
UBE4B: A Promising Regulatory Molecule in Neuronal Death and Survival
Previous Article in Special Issue
Flavin-Dependent Enzymes in Cancer Prevention
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

1,†
,
2,†
,
3
,
2
 and
1,3,*
1 Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy 2 Department of Cellular Biology, University of Calabria, via Bucci 4c, I-87036, Arcavacata di Rende, Italy 3 Institute of Biomembranes and Bioenergetics, CNR, via Amendola 165/A, I-70126, Bari, Italy These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 10 October 2012 / Revised: 14 November 2012 / Accepted: 20 November 2012 / Published: 11 December 2012
(This article belongs to the Special Issue Flavins)
View Full-Text   |   Download PDF [1119 KB, uploaded 19 June 2014]   |   Browse Figures
SciFeed

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 FLAD1; human FAD synthase; FMN adenylyltransferase; Flavin; FAD; PAPS reductase domain; molybdopterin-binding domain
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

Share & Cite This Article

Further Mendeley | CiteULike
Export to BibTeX |
EndNote |
RIS
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.

View more citation formats

Related Articles

Article Metrics

For more information on the journal, click here

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert