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Literature summary 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 PubMed

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.26 D168A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 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 Nakaseomyces glabratus
2.7.1.26 D66A site-directed mutagenesis, inactive mutant Nakaseomyces glabratus
2.7.1.26 N62A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 N62S site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 R297A site-directed mutagenesis, the mutant shows a 2fold increased activity compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 R297A/R300A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 R300A site-directed mutagenesis, the mutant shows 93% reduced activity compared to the wild-type enzyme Nakaseomyces glabratus
2.7.1.26 W184A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 D168A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus
2.7.7.2 D66A site-directed mutagenesis, inactive mutant Nakaseomyces glabratus
2.7.7.2 N62A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 N62S site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 R297A site-directed mutagenesis, the mutant shows a 2fold increased activity compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 R297A/R300A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 R300A site-directed mutagenesis, the mutant shows 93% reduced activity compared to the wild-type enzyme Nakaseomyces glabratus
2.7.7.2 W184A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Nakaseomyces glabratus

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.1.26 FAD product inhibition, wild-type enzyme FMNAT is strongly inhibited by FAD, whereas D181A mutant enzyme has an attenuated product inhibition Nakaseomyces glabratus
2.7.7.2 FAD product inhibition, wild-type enzyme FMNAT is strongly inhibited by FAD, whereas D181A mutant enzyme has an attenuated product inhibition Nakaseomyces glabratus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.1.26 additional information
-
 additional information steady-state kinetic analysis of wild-type and mutant enzymes Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus
2.7.7.2 0.0005
-
 FMN pH and temperature not specified in the publication, mutant N62A Nakaseomyces glabratus
2.7.7.2 0.001
-
 FMN pH and temperature not specified in the publication, wild-type enzyme Nakaseomyces glabratus
2.7.7.2 0.0011
-
 FMN pH and temperature not specified in the publication, mutant N62S Nakaseomyces glabratus
2.7.7.2 0.0015
-
 FMN pH and temperature not specified in the publication, mutant D181A Nakaseomyces glabratus
2.7.7.2 0.0023
-
 FMN pH and temperature not specified in the publication, mutant R300A Nakaseomyces glabratus
2.7.7.2 0.003
-
 FMN pH and temperature not specified in the publication, mutant R297A Nakaseomyces glabratus
2.7.7.2 0.0071
-
 FMN pH and temperature not specified in the publication, mutant D168A Nakaseomyces glabratus
2.7.7.2 0.0093
-
 FMN pH and temperature not specified in the publication, mutant R297A/R300A Nakaseomyces glabratus
2.7.7.2 0.1994
-
 FMN pH and temperature not specified in the publication, mutant W184A Nakaseomyces glabratus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.26 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 Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.26 Nakaseomyces glabratus Q6FNA9
-
-
2.7.7.2 Nakaseomyces glabratus Q6FNA9
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
2.7.7.2 ATP + FMN = diphosphate + FAD product release may be the rate-limiting step of the reaction Nakaseomyces glabratus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.26 ATP + riboflavin
-
 Nakaseomyces glabratus ADP + FMN
-
 ?
2.7.1.26 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 Nakaseomyces glabratus ?
-
 ?
2.7.1.26 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 Nakaseomyces glabratus ?
-
 ?
2.7.7.2 ATP + FMN
-
 Nakaseomyces glabratus diphosphate + FAD
-
 ?
2.7.7.2 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 Nakaseomyces glabratus ?
-
 ?
2.7.7.2 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 Nakaseomyces glabratus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.1.26 additional information 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 Nakaseomyces glabratus
2.7.7.2 additional information 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 Nakaseomyces glabratus

Synonyms

EC Number Synonyms Comment Organism
2.7.1.26 FMN adenylyltransferase
-
 Nakaseomyces glabratus
2.7.1.26 FMNAT
-
 Nakaseomyces glabratus
2.7.7.2 FMN adenylyltransferase
-
 Nakaseomyces glabratus
2.7.7.2 FMNAT
-
 Nakaseomyces glabratus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.2 0.01
-
 FMN pH and temperature not specified in the publication, mutant R300A Nakaseomyces glabratus
2.7.7.2 0.012
-
 FMN pH and temperature not specified in the publication, mutant R297A/R300A Nakaseomyces glabratus
2.7.7.2 0.017
-
 FMN pH and temperature not specified in the publication, mutant N62S Nakaseomyces glabratus
2.7.7.2 0.017
-
 FMN pH and temperature not specified in the publication, mutant D168A Nakaseomyces glabratus
2.7.7.2 0.042
-
 FMN pH and temperature not specified in the publication, mutant N62A Nakaseomyces glabratus
2.7.7.2 0.089
-
 FMN pH and temperature not specified in the publication, wild-type enzyme Nakaseomyces glabratus
2.7.7.2 0.21
-
 FMN pH and temperature not specified in the publication, mutant R297A Nakaseomyces glabratus
2.7.7.2 0.26
-
 FMN pH and temperature not specified in the publication, mutant W184A Nakaseomyces glabratus
2.7.7.2 0.88
-
 FMN pH and temperature not specified in the publication, mutant D181A Nakaseomyces glabratus

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.26 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 Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.7.7.2 0.01
-
 FAD versus ATP, pH and temperature not specified in the publication, wild-type enzyme Nakaseomyces glabratus
2.7.7.2 0.012
-
 FAD versus FMN, pH and temperature not specified in the publication, wild-type enzyme Nakaseomyces glabratus

General Information

EC Number General Information Comment Organism
2.7.1.26 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 Nakaseomyces glabratus
2.7.1.26 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 Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus
2.7.7.2 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 Nakaseomyces glabratus