Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.
FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy. Electronic address: teresaanna.giancaspero@uniba.it.
Teresa Anna
TA
Giancaspero
Dipartimento DiBEST, Biologia, Ecologia, Scienze della Terra, Unità di Biochimica e Biotecnologie Molecolari, Università della Calabria, via Bucci 4c, I-87036, Arcavacata di Rende, Italy. Electronic address: mgalluccio@unical.it.
Michele
M
Galluccio
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy. Electronic address: miccolis.angelica@libero.it.
Angelica
A
Miccolis
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy. Electronic address: pieroleone87@gmail.com.
Piero
P
Leone
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Balzaretti 9, I-20133, Milano, Italy. Electronic address: ivano.eberini@unimi.it.
Ivano
I
Eberini
Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, via Celoria 2, I-20133, Milano, Italy. Electronic address: stefania.iametti@unimi.it.
Stefania
S
Iametti
Dipartimento DiBEST, Biologia, Ecologia, Scienze della Terra, Unità di Biochimica e Biotecnologie Molecolari, Università della Calabria, via Bucci 4c, I-87036, Arcavacata di Rende, Italy. Electronic address: cesare.indiveri@unical.it.
Cesare
C
Indiveri
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy. Electronic address: maria.barile@uniba.it.
Maria
M
Barile
D000595:Amino Acid Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D003198:Computer Simulation; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011621:Pteridines / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003067:Coenzymes; D005182:Flavin-Adenine Dinucleotide; D006867:Hydrolases; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes; D009713:Nucleotidyltransferases; D011621:Pteridines; C022261:molybdopterin; C020143:FMN adenylyltransferase
Cysteine; FAD hydrolase; FAD pyrophosphatase; Human FAD synthase; Molybdopterin-binding domain; Redox switch