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Literature summary for 4.1.99.26 extracted from

  • Ayikpoe, R.; Ngendahimana, T.; Langton, M.; Bonitatibus, S.; Walker, L.; Eaton, S.; Eaton, G.; Pandelia, M.; Elliott, S.; Latham, J.
    Spectroscopic and electrochemical characterization of the mycofactocin biosynthetic protein, MftC, provides insight into its redox flipping mechanism (2019), Biochemistry, 58, 940-950 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 4.1.99.26

Cloned(Commentary)

Cloned (Comment) Organism
gene mftC, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Bl21(DE3) Mycobacterium ulcerans

Protein Variants

Protein Variants Comment Organism
C251A insoluble Mycobacterium ulcerans
C251A site-directed mutagenesis, the mutant is insoluble Mycobacterium ulcerans
C258A site-directed mutagenesis, the mutant shows no altered phenotype Mycobacterium ulcerans
C269A auxiliary [4Fe-4S] cluster I mutant, capable of catalyzing the reductive cleavage of SAM to form dAdo but incapable of converting MftA to MftA* or MftA** Mycobacterium ulcerans
C269A site-directed mutagenesis, an Aux I KO variant mutant, the mutant is soluble Mycobacterium ulcerans
C269A/C323A insoluble Mycobacterium ulcerans
C269A/C323A site-directed mutagenesis, an Aux I KO variant mutant, the mutant is insoluble Mycobacterium ulcerans
C30A/C34A/C37A site-directed mutagenesis, insoluble mutant Mycobacterium ulcerans
C30A/C34A/C37A insoluble Mycobacterium ulcerans
C30A/C37A radical S-adenosylmethionine mutant, can neither cleave SAM nor modify MftA Mycobacterium ulcerans
C30A/C37A site-directed mutagenesis, a RS KO variant double mutant Mycobacterium ulcerans
C310A site-directed mutagenesis, an Aux II KO variant mutant Mycobacterium ulcerans
C310A/C341A auxiliary [4Fe-4S] cluster II mutant, capable of catalyzing the reductive cleavage of SAM to form dAdo, incapable of converting MftA to MftA* or MftA** Mycobacterium ulcerans
C310A/C341A site-directed mutagenesis, an Aux II KO variant double mutant Mycobacterium ulcerans
C323A auxiliary [4Fe-4S] cluster I mutant, capable of catalyzing the reductive cleavage of SAM to form dAdo, incapable of converting MftA to MftA* or MftA** Mycobacterium ulcerans
C323A site-directed mutagenesis, an Aux I KO variant mutant, the mutant is soluble Mycobacterium ulcerans
C341A site-directed mutagenesis, an Aux II KO variant mutant Mycobacterium ulcerans
additional information EPR analysis of MftC variants with substrate bound providing insight to the function of the [Fe-S] clusters Mycobacterium ulcerans
additional information systematical replacement of Cys residues by Ala. The RS KO could neither cleave SAM nor modify MftA, consistent with the successful knockout of the RS cluster. Activity assays for Aux I and Aux II KO's also provided insightful results. Both Aux I and Aux II KO's were capable of catalyzing the reductive cleavage of SAM to form dAdo (Figure 3A), suggesting that the RS cluster remained intact and in an active conformation in the mutated proteins. However, when assayed against MftA, both Aux I and Aux II KO's are incapable of converting MftA to MftA* or MftA** Mycobacterium ulcerans

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ in a [4Fe-4S] clusters, all three [Fe-S] clusters are required for MftC modification of substrate MftA Mycobacterium ulcerans
additional information iron and sulfur quantification, identification of conserved cysteines in MftC, overview Mycobacterium ulcerans

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
42000
-
 recombinant His-tagged enzyme, gel filtration Mycobacterium ulcerans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
C-terminal [mycofactocin precursor peptide MftA]-glycyl-3-amino-5-[(4-hydroxyphenyl)methyl]-4,4-dimethylpyrrolidin-2-one + 5'-deoxyadenosine + L-methionine + A Mycobacterium ulcerans
-
 C-terminal [mycofactocin precursor peptide]-glycyl-L-valyl-4-[2-aminoethenyl]phenol + S-adenosyl-L-methionine + AH2
-
 ?
C-terminal [mycofactocin precursor peptide]-glycyl-L-valyl-4-[2-aminoethenyl]phenol + S-adenosyl-L-methionine + AH2 Mycobacterium ulcerans
-
 C-terminal [mycofactocin precursor peptide]-glycyl-3-amino-5-[(4-hydroxyphenyl)methyl]-4,4-dimethylpyrrolidin-2-one + 5'-deoxyadenosine + L-methionine + A
-
 ?
additional information Mycobacterium ulcerans enzyme additionally catalyzes the reaction of MftA peptide to MftA carrying the alpha/beta unsaturated bond, i.e. MftA**, reaction of EC 1.3.98.7 ?
-
-
additional information Mycobacterium ulcerans Agy99 enzyme additionally catalyzes the reaction of MftA peptide to MftA carrying the alpha/beta unsaturated bond, i.e. MftA**, reaction of EC 1.3.98.7 ?
-
-

Organism

Organism UniProt Comment Textmining
Mycobacterium ulcerans A0PM49
-
-
Mycobacterium ulcerans A0PM49 cf. EC 1.3.98.7
-
Mycobacterium ulcerans Agy99 A0PM49
-
-
Mycobacterium ulcerans Agy99 A0PM49 cf. EC 1.3.98.7
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) by nickel affinity chromatography to near homogeneity Mycobacterium ulcerans

Reaction

Reaction Comment Organism Reaction ID
C-terminal [mycofactocin precursor peptide]-glycyl-3-amino-5-[(4-hydroxyphenyl)methyl]-4,4-dimethylpyrrolidin-2-one + 5'-deoxyadenosine + L-methionine + A = C-terminal [mycofactocin precursor peptide]-glycyl-L-valyl-4-[2-aminoethenyl]phenol + S-adenosyl-L-methionine + AH2 enzyme mechanism, overview Mycobacterium ulcerans
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
C-terminal [mycofactocin precursor peptide MftA]-glycyl-3-amino-5-[(4-hydroxyphenyl)methyl]-4,4-dimethylpyrrolidin-2-one + 5'-deoxyadenosine + L-methionine + A
-
 Mycobacterium ulcerans C-terminal [mycofactocin precursor peptide]-glycyl-L-valyl-4-[2-aminoethenyl]phenol + S-adenosyl-L-methionine + AH2
-
 ?
C-terminal [mycofactocin precursor peptide]-glycyl-L-valyl-4-[2-aminoethenyl]phenol + S-adenosyl-L-methionine + AH2
-
 Mycobacterium ulcerans C-terminal [mycofactocin precursor peptide]-glycyl-3-amino-5-[(4-hydroxyphenyl)methyl]-4,4-dimethylpyrrolidin-2-one + 5'-deoxyadenosine + L-methionine + A
-
 ?
additional information enzyme additionally catalyzes the reaction of MftA peptide to MftA carrying the alpha/beta unsaturated bond, i.e. MftA**, reaction of EC 1.3.98.7 Mycobacterium ulcerans ?
-
-
additional information MftC catalyzes the cleavage of SAM to form dAdo and it converts MftA to MftA* Mycobacterium ulcerans ?
-
-
additional information enzyme additionally catalyzes the reaction of MftA peptide to MftA carrying the alpha/beta unsaturated bond, i.e. MftA**, reaction of EC 1.3.98.7 Mycobacterium ulcerans Agy99 ?
-
-

Synonyms

Synonyms Comment Organism
mftC
-
 Mycobacterium ulcerans
mycofactocin biosynthetic protein
-
 Mycobacterium ulcerans
mycofactocin maturase
-
 Mycobacterium ulcerans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Mycobacterium ulcerans

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine MtfC is a radical S-adenosylmethionine (RS, SAM) protein Mycobacterium ulcerans
S-adenosyl-L-methionine residues Cys30, 34, and 37 are the radical S-adenosylmethionine ligands Mycobacterium ulcerans
[4Fe-4S] cluster an iron-sulfur protein Mycobacterium ulcerans
[4Fe-4S]-center MftC binds a radical S-adenosylmethionine [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Presence of S-adenosylmethionine and MftA affects the environments of the radical S-adenosylmethionine and Aux I cluster whereas the Aux II cluster is unaffected by the substrates. All three cluster are required for cataylsis Mycobacterium ulcerans

General Information

General Information Comment Organism
evolution the enzyme belongs to a subset of the RS proteins that modify peptides belong to the SPASM subfamily (subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin) and are annotated as RS-SPASM proteins. RS-SPASM proteins are comprised of a prototypical TIM barrel fold which binds the RS [4Fe-4S] cluster involved in the homolytic cleavage of S-adenosylmethionine (SAM). In addition, they contain an elongated C-terminal SPASM domain which binds up to two additional auxiliary [Fe-S] clusters. The cluster proximal to the RS cluster (annotated as Aux I) is typically a [4Fe-4S] (with the exception of PqqE in which a [2Fe-2S] cluster is reported) and the distal cluster (designated as Aux II) is a [4Fe-4S] cluster. MftC is a RS-SPASM enzyme that performs the first modifying step in the biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), mycofactocin. The ability of MftC to redox-flip, or accommodate both oxidative to redox neutral reactions, is certainly unique to the RS-SPASM subfamily. The Aux I and Aux II clusters are required for catalysis, consistent with results shown for QhpD, AnSME, SCIFF, and PqqE Mycobacterium ulcerans
malfunction the enzyme knockout (KO) mutant can neither cleave SAM nor modify MftA, consistent with the successful knockout of the RS cluster. Both Aux I and Aux II KO's are capable of catalyzing the reductive cleavage of SAM to form dAdo, suggesting that the RS cluster remains intact and in an active conformation in the mutated proteins. When assayed against MftA, both Aux I and Aux II KO's are incapable of converting MftA to MftA* or MftA** Mycobacterium ulcerans
metabolism following MftC modification, MftE hydrolyzes 3-amino-5-[(p-hydroxyphenyl) methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP) moiety (MftA*) at the valine position, freeing AHDP from the peptide Mycobacterium ulcerans
additional information MtfC homology structure modelling using the structures of anSME (PDB ID 4K36), CteB (PDB ID 5WHY), or SuiB (PDB ID 5V1T) as templates. Identification of conserved cysteines in MftC, overview. The Aux I and Aux II clusters are required for catalysis Mycobacterium ulcerans
physiological function MftC is a RS-SPASM enzyme that performs the first modifying step in the biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), mycofactocin. The mycofactocin maturase, MftC, is a unique radical S-adenosylmethionine (RS, SAM) protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA Mycobacterium ulcerans