FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3′-phosphoadenosine-5′- phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated networkamong the Cys residues in this domain. In thismodel, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399–C402, C303–C312, both in the PAPS domain), and two unstable disulfides (C39–C50; C440–C464).Whereas the C39–C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

Significance of redox-active cysteines in human FAD synthase isoform 2 / A. Miccolis, M. Galluccio, C. Nitride, T.A. Giancaspero, P. Ferranti, S. Iametti, C. Indiveri, F. Bonomi, M. Barile. - In: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS. - ISSN 1570-9639. - 1844:12(2014 Dec), pp. 2086-2095. [10.1016/j.bbapap.2014.08.005]

Significance of redox-active cysteines in human FAD synthase isoform 2

S. Iametti;F. Bonomi
Penultimo
;
2014

Abstract

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3′-phosphoadenosine-5′- phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated networkamong the Cys residues in this domain. In thismodel, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399–C402, C303–C312, both in the PAPS domain), and two unstable disulfides (C39–C50; C440–C464).Whereas the C39–C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.
Human FAD synthase; FMN adenylyltransferase; Thiol; Disulfide bridge; Redox sensing; Mercury toxicity
Settore BIO/10 - Biochimica
Article (author)
File in questo prodotto:
File Dimensione Formato  
1-s2.0-S1570963914002039-main.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 2.27 MB
Formato Adobe PDF
2.27 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/253043
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 15
  • ???jsp.display-item.citation.isi??? 15
social impact