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Literature summary extracted from

  • Iamurri, S.M.; Daugherty, A.B.; Edmondson, D.E.; Lutz, S.
    Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides (2013), Protein Eng. Des. Sel., 26, 791-795.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.7.1.26 synthesis the enzyme is used for the preparation of flavin mononucleotide (FMN) and FMN analogues from their corresponding riboflavin precursors, which is performed in a two-step procedure. After initial enzymatic conversion of riboflavin to FAD by the bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. The engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale Corynebacterium ammoniagenes
2.7.7.2 synthesis the enzyme is used for the preparation of flavin mononucleotide (FMN) and FMN analogues from their corresponding riboflavin precursors, which is performed in a two-step procedure. After initial enzymatic conversion of riboflavin to FAD by the bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. The engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale Corynebacterium ammoniagenes

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.26 gene ribF, recombinant expression of C-terminally poly-His-tagged N-terminally truncated mutant in Escherichia coli Corynebacterium ammoniagenes
2.7.7.2 recombinant expression of C-terminally poly-His-tagged N-terminally truncated mutant in Escherichia coli Corynebacterium ammoniagenes

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.26 additional information engineering of the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain leads to a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale. Deletion of the N-terminal adenosyl transfer domain in the truncated C-terminal RF kinase domain, tcRFK, variants results in a drop in the TM value from 40°C (parental CaFADS) to 35°C for tcRFK. Addition of the C-terminal poly-His tag further reduces the TM to 30°C, presumably due to the conformationally flexible tail formed by the extra amino acids Corynebacterium ammoniagenes
2.7.7.2 additional information engineering of the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain leads to a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale. Deletion of the N-terminal adenosyl transfer domain in the truncated C-terminal RF kinase domain, tcRFK, variants results in a drop in the TM value from 40°C (parental CaFADS) to 35°C for tcRFK. Addition of the C-terminal poly-His tag further reduces the TM to 30°C, presumably due to the conformationally flexible tail formed by the extra amino acids Corynebacterium ammoniagenes

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.26 Mg2+ required Corynebacterium ammoniagenes
2.7.7.2 Mg2+ required Corynebacterium ammoniagenes

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.1.26 additional information
-
 the elution profile of full-length FADS shows two characteristic peaks at molecular weights corresponding to its monomeric and trimeric forms, while the tcRFK elutes as a single peak at an estimated molecular weight of 16.6 kDa, consistent with monomeric enzyme Corynebacterium ammoniagenes
2.7.1.26 16600
-
 recombinant truncated C-terminal RF kinase domain, gel filtration Corynebacterium ammoniagenes
2.7.7.2 additional information
-
 the elution profile of full-length FADS shows two characteristic peaks at molecular weights corresponding to its monomeric and trimeric forms, while the tcRFK elutes as a single peak at an estimated molecular weight of 16.6 kDa, consistent with monomeric enzyme Corynebacterium ammoniagenes
2.7.7.2 16600
-
 recombinant truncated C-terminal RF kinase domain, gel filtration Corynebacterium ammoniagenes

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 Corynebacterium ammoniagenes
-
 ADP + FMN
-
 ?
2.7.1.26 additional information Corynebacterium ammoniagenes 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 Corynebacterium ammoniagenes engineered mutant diphosphate + FAD
-
 ?
2.7.7.2 additional information Corynebacterium ammoniagenes 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 Corynebacterium ammoniagenes Q59263 gene ribF
-
2.7.7.2 Corynebacterium ammoniagenes Q59263 gene ribF
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.26 recombinant C-terminally poly-His-tagged N-terminally truncated mutant_187-338 from Escherichia coli by ion exchange chromatography and gel filtration Corynebacterium ammoniagenes
2.7.7.2 recombinant C-terminally poly-His-tagged N-terminally truncated mutant_187-338 from Escherichia coli by ion exchange chromatography and gel filtration Corynebacterium ammoniagenes

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.26 ATP + 5-deazariboflavin
-
 Corynebacterium ammoniagenes ADP + 5-deazariboflavin monophosphate
-
 ?
2.7.1.26 ATP + 7,8-dichlororiboflavin
-
 Corynebacterium ammoniagenes ADP + 7,8-dichlororiboflavin monophosphate
-
 ?
2.7.1.26 ATP + 8-aminoriboflavin
-
 Corynebacterium ammoniagenes ADP + 8-aminoriboflavin monophosphate
-
 ?
2.7.1.26 ATP + riboflavin
-
 Corynebacterium ammoniagenes 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 Corynebacterium ammoniagenes ?
-
 ?
2.7.1.26 additional information the engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale, method evaluation, overview Corynebacterium ammoniagenes ?
-
 ?
2.7.7.2 ATP + FMN engineered mutant Corynebacterium ammoniagenes 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 Corynebacterium ammoniagenes ?
-
 ?
2.7.7.2 additional information the engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale, method evaluation, overview Corynebacterium ammoniagenes ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.1.26 monomer x * 16600, recombinant truncated C-terminal RF kinase domain, SDS-PAGE Corynebacterium ammoniagenes
2.7.7.2 monomer x * 16600, recombinant truncated C-terminal RF kinase domain, SDS-PAGE Corynebacterium ammoniagenes

Synonyms

EC Number Synonyms Comment Organism
2.7.1.26 RFK
-
 Corynebacterium ammoniagenes
2.7.7.2 FADS
-
 Corynebacterium ammoniagenes

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.26 ATP
-
 Corynebacterium ammoniagenes
2.7.7.2 ATP
-
 Corynebacterium ammoniagenes