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Literature summary for 2.7.7.2 extracted from

  • Huerta, C.; Grishin, N.V.; Zhang, H.
    The super mutant of yeast FMN adenylyltransferase enhances the enzyme turnover rate by attenuating product inhibition (2013), Biochemistry, 52, 3615-3617.
    View publication on PubMedView publication on EuropePMC

Protein Variants

Protein Variants Comment Organism
D168A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme [Candida] glabrata
D181A site-directed mutagenesis, the mutant shows reduced sensitivity to inhibition by FAD compared to the wild-type enzyme and has a much faster turnover rate than the wild-type enzyme [Candida] glabrata
D66A site-directed mutagenesis, inactive mutant [Candida] glabrata
N62A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme [Candida] glabrata
N62S site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme [Candida] glabrata
R297A site-directed mutagenesis, the mutant shows a 2fold increased activity compared to the wild-type enzyme [Candida] glabrata
R297A/R300A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme [Candida] glabrata
R300A site-directed mutagenesis, the mutant shows 93% reduced activity compared to the wild-type enzyme [Candida] glabrata
W184A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme [Candida] glabrata

Inhibitors

Inhibitors Comment Organism Structure
FAD product inhibition, wild-type enzyme FMNAT is strongly inhibited by FAD, whereas D181A mutant enzyme has an attenuated product inhibition [Candida] glabrata

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic analysis of wild-type and mutant enzymes. The enzyme from Candida glabrata apparently binds its substrates with high affinity, but the overall turnover rate is very slow due to product inhibition [Candida] glabrata
0.0005
-
FMN pH and temperature not specified in the publication, mutant N62A [Candida] glabrata
0.001
-
FMN pH and temperature not specified in the publication, wild-type enzyme [Candida] glabrata
0.0011
-
FMN pH and temperature not specified in the publication, mutant N62S [Candida] glabrata
0.0015
-
FMN pH and temperature not specified in the publication, mutant D181A [Candida] glabrata
0.0023
-
FMN pH and temperature not specified in the publication, mutant R300A [Candida] glabrata
0.003
-
FMN pH and temperature not specified in the publication, mutant R297A [Candida] glabrata
0.0071
-
FMN pH and temperature not specified in the publication, mutant D168A [Candida] glabrata
0.0093
-
FMN pH and temperature not specified in the publication, mutant R297A/R300A [Candida] glabrata
0.1994
-
FMN pH and temperature not specified in the publication, mutant W184A [Candida] glabrata

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required, the interaction with the two Mg2+ ion coordinating waters by Asp168 is important for the catalytic activity of the enzym. Residues Asn62, Asp66, Asp168, and Arg297 interact either with ATP phosphate groups, or coordinate the catalytic Mg2+ ion either directly or indirectly through water moleculese [Candida] glabrata

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + FMN [Candida] glabrata
-
diphosphate + FAD
-
?
additional information [Candida] glabrata bifunctional FAD synthetase exhibiting both the activities of FAD synthetase, EC 2.7.7.2, and riboflavin kinase, EC 2.7.1.26 ?
-
?

Organism

Organism UniProt Comment Textmining
[Candida] glabrata Q6FNA9
-
-

Reaction

Reaction Comment Organism Reaction ID
ATP + FMN = diphosphate + FAD product release may be the rate-limiting step of the reaction [Candida] glabrata

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + FMN
-
[Candida] glabrata diphosphate + FAD
-
?
additional information bifunctional FAD synthetase exhibiting both the activities of FAD synthetase, EC 2.7.7.2, and riboflavin kinase, EC 2.7.1.26 [Candida] glabrata ?
-
?
additional information the flavin motif is involved in flavin ligand binding, role of active site residues in the catalytic mechanism, overview. The isoalloxazine ring is sandwiched between the indole ring of Trp184 and the planar guanidinium group of Arg189, while the hydrophilic pyrimidine ring forms two specific hydrogen bonds between its C4 carbonyl and the main chain amide of Asp181, and between its N3 amide and the side chain of Asp181, respectively. Residues Asn62, Asp66, Asp168, and Arg297 interact either with ATP phosphate groups, or to coordinate the catalytic Mg2+ ion either directly or indirectly through water molecules. Arg297 might be involved in the interaction with the phosphate groups of both substrates, and helps in their positioning for the nucleophilic attack in the adenylyltransfer reaction [Candida] glabrata ?
-
?

Subunits

Subunits Comment Organism
More the enzyme contains a core domain with a modified Rossman-fold topology and a C-terminal extension. The substrate binding and catalytic site is located at the interface of the two domains [Candida] glabrata

Synonyms

Synonyms Comment Organism
FMN adenylyltransferase
-
[Candida] glabrata
FMNAT
-
[Candida] glabrata

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.01
-
FMN pH and temperature not specified in the publication, mutant R300A [Candida] glabrata
0.012
-
FMN pH and temperature not specified in the publication, mutant R297A/R300A [Candida] glabrata
0.017
-
FMN pH and temperature not specified in the publication, mutant D168A [Candida] glabrata
0.017
-
FMN pH and temperature not specified in the publication, mutant N62S [Candida] glabrata
0.042
-
FMN pH and temperature not specified in the publication, mutant N62A [Candida] glabrata
0.089
-
FMN pH and temperature not specified in the publication, wild-type enzyme [Candida] glabrata
0.21
-
FMN pH and temperature not specified in the publication, mutant R297A [Candida] glabrata
0.26
-
FMN pH and temperature not specified in the publication, mutant W184A [Candida] glabrata
0.88
-
FMN pH and temperature not specified in the publication, mutant D181A [Candida] glabrata

Cofactor

Cofactor Comment Organism Structure
ATP residues Asn62, Asp66, Asp168, and Arg297 interact either with ATP phosphate groups, or coordinate the catalytic Mg2+ ion either directly or indirectly through water moleculese [Candida] glabrata

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.01
-
FAD versus ATP, pH and temperature not specified in the publication, wild-type enzyme [Candida] glabrata
0.012
-
FAD versus FMN, pH and temperature not specified in the publication, wild-type enzyme [Candida] glabrata

General Information

General Information Comment Organism
evolution eukaryotic FMNAT is related to phosphoadenosine phosphosulfate (PAPS) reductase family proteins and contains a core domain with a modified Rossman-fold topology and a C-terminal extension [Candida] glabrata
physiological function flavocoenzymes, including flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are versatile redox cofactors involved in many fundamental cellular processes in all living organisms. FAD is synthesized from riboflavin obtained from the diet via two enzymatic steps catalyzed by riboflavin kinase (RFK, EC 2.7.1.26) and essential FMN adenylyltransferase (FMNAT,EC 2.7.7.2). Phosphorylation of riboflavin by RFK is crucial for specific absorption of the vitamin and is the physiologically rate-limiting step in the biosynthesis of flavocoenzymes, whereas product (FAD) feedback inhibition is observed for mammalian FMNAT, suggesting that biosynthesis of FAD is also regulated at the FMNAT reaction step [Candida] glabrata