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FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
FAD + L-threonyl-[Shewanella oneidensis NqrC protein]
AMP + FMN-L-threonyl-[Shewanella oneidensis NqrC protein] + H+
a high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identifies an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins
-
-
?
FAD + [Klebsiella pneumoniae cytoplasmic fumarate reductase]-L-threonine
[Klebsiella pneumoniae cytoplasmic fumarate reductase]-FMN-L-threonine + AMP
-
-
-
?
FAD + [NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase]-L-threonine
[NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase]-FMN-L-threonine + AMP
-
-
-
?
FAD + [periplasmic redox-carrying protein RnfG_Ec]-L-threonine
[periplasmic redox-carrying protein RnfG_Ec]-FMN-L-threonine + AMP
flavinylating via the metal-dependent covalent attachment of FMN
-
-
?
FAD + [protein NqrC]-L-threonine
[protein NqrC]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-serine
[protein]-FMN-L-serine + AMP
45-90% activity compared to threonine as acceptor with wild-type and mutant enzymes
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
FAD + [Shewanella oneidensis protein NqrC]-L-threonine
[Shewanella oneidensis protein NqrC]-FMN-L-threonine + AMP
a subunit (NqrC) of a cytoplasmic membrane redox system (Nqr), detection of an essential lysine residue in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoprotein
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-
?
FAD + [Vibrio cholerae protein NqrC]-L-threonine
[Vibrio cholerae protein NqrC]-FMN-L-threonine + AMP
additional information
?
-
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[NqrC protein]
AMP + FMN-L-threonyl-[NqrC protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
RnfG_Ec subunit of the Rnf_Ec-redox system of Escherichia coli
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-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
RnfG_Ec subunit of the Rnf_Ec-redox system of Escherichia coli
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-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
-
-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
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-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
-
-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
-
-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
-
-
?
FAD + L-threonyl-[RnfG protein]
AMP + FMN-L-threonyl-[RnfG protein] + H+
RnfG subunit of the Rnf redox system
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
preferred substrate
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
-
?
FAD + [Vibrio cholerae protein NqrC]-L-threonine
[Vibrio cholerae protein NqrC]-FMN-L-threonine + AMP
-
-
-
?
FAD + [Vibrio cholerae protein NqrC]-L-threonine
[Vibrio cholerae protein NqrC]-FMN-L-threonine + AMP
conserved Lys207 in NqrC can account for the pH dependency
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-
?
additional information
?
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD and its covalent binding to the hydroxyl group of a threonine residue in a target flavoprotein (EC 2.7.1.180). The enzyme is capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A single amino acid substitution Y60N converts it from an FAD-binding protein to a Mg2+-dependent FAD diphosphatase (Ftp_Tp-like) (EC 3.6.1.18). The engineered protein variant (Ftp_EcY60A) shows Mg2+-dependent FAD diphosphatase activity, but also retains its Mg2+-dependent FMN transferase (EC 2.7.1.180) activity on the protein substrate, indicating that the protein variant enzyme has dual activity. The Ftp_EcY60A protein variant binds FAD, yet rapidly hydrolyzes it and the product FMN dissociates. Substrate binding structures, detailed overview
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-
additional information
?
-
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD and its covalent binding to the hydroxyl group of a threonine residue in a target flavoprotein (EC 2.7.1.180). The enzyme is capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A single amino acid substitution Y60N converts it from an FAD-binding protein to a Mg2+-dependent FAD diphosphatase (Ftp_Tp-like) (EC 3.6.1.18). The engineered protein variant (Ftp_EcY60A) shows Mg2+-dependent FAD diphosphatase activity, but also retains its Mg2+-dependent FMN transferase (EC 2.7.1.180) activity on the protein substrate, indicating that the protein variant enzyme has dual activity. The Ftp_EcY60A protein variant binds FAD, yet rapidly hydrolyzes it and the product FMN dissociates. Substrate binding structures, detailed overview
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additional information
?
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the Escherichia coli enzyme shows no Mg2+-dependent FAD pyrophosphatase (EC 3.6.1.18) activity
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additional information
?
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the Escherichia coli enzyme shows no Mg2+-dependent FAD pyrophosphatase (EC 3.6.1.18) activity
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additional information
?
-
Klebsiella pneumoniae ApbE can modify both threonine and serine residues in the position 447 of FRD resulting in their similar activities, although serine is modified less readily
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-
additional information
?
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-
Klebsiella pneumoniae ApbE can modify both threonine and serine residues in the position 447 of FRD resulting in their similar activities, although serine is modified less readily
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-
additional information
?
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD and its covalent binding to the hydroxyl group of a threonine residue in a target flavoprotein (EC 2.7.1.180). The enzyme is capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. It also displays FAD diphosphatase activity in vitro, hydrolyzing FAD into FMN and AMP (EC 3.6.1.18). Substrate binding structures, detailed overview
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-
-
additional information
?
-
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD and its covalent binding to the hydroxyl group of a threonine residue in a target flavoprotein (EC 2.7.1.180). The enzyme is capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. It also displays FAD diphosphatase activity in vitro, hydrolyzing FAD into FMN and AMP (EC 3.6.1.18). Substrate binding structures, detailed overview
-
-
-
additional information
?
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD and its covalent binding to the hydroxyl group of a threonine residue in a target flavoprotein (EC 2.7.1.180). The enzyme is capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. It also displays FAD diphosphatase activity in vitro, hydrolyzing FAD into FMN and AMP (EC 3.6.1.18). Substrate binding structures, detailed overview
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-
additional information
?
-
substrate specificity, overview. FAD cannot be substituted by FMN
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-
additional information
?
-
the His257 residue plays important roles in catalysis and in enzyme-substrate complex formation and in substrate deprotonation, proposed mechanism of flavin transfer
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-
additional information
?
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-
the His257 residue plays important roles in catalysis and in enzyme-substrate complex formation and in substrate deprotonation, proposed mechanism of flavin transfer
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-
additional information
?
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-
no activity with FMN
-
-
?
additional information
?
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
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?
additional information
?
-
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
-
-
?
additional information
?
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
-
-
?
additional information
?
-
-
no activity with FMN
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-
?
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FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
FAD + [Klebsiella pneumoniae cytoplasmic fumarate reductase]-L-threonine
[Klebsiella pneumoniae cytoplasmic fumarate reductase]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
FAD + [Vibrio cholerae protein NqrC]-L-threonine
[Vibrio cholerae protein NqrC]-FMN-L-threonine + AMP
-
-
-
?
additional information
?
-
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + L-threonyl-[protein]
AMP + FMN-L-threonyl-[protein] + H+
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
-
?
FAD + [protein]-L-threonine
[protein]-FMN-L-threonine + AMP
-
-
-
-
?
additional information
?
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
-
-
?
additional information
?
-
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
-
-
?
additional information
?
-
enzyme ApbE covalently attaches flavin residues to threonine residues of Na+-translocating NADH:quinone oxidoreductase maturation, Na+-NQR
-
-
?
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0.011 - 0.119
[protein NqrC]-L-threonine
-
0.1
[Vibrio cholerae protein NqrC]-L-threonine
above, pH 9.0, 22°C, recombinant wild-type enzyme
-
additional information
additional information
-
0.00006
FAD
pH 9.0, 22°C, recombinant wild-type enzyme
0.0001
FAD
pH 9.0, temperature not specified in the publication, wild-type enzyme
0.00019
FAD
pH 9.0, temperature not specified in the publication, mutant H257t
0.0002
FAD
pH 9.0, temperature not specified in the publication, mutant H257G
0.011
[protein NqrC]-L-threonine
pH 9.0, temperature not specified in the publication, wild-type enzyme
-
0.087
[protein NqrC]-L-threonine
pH 9.0, temperature not specified in the publication, mutant H257G
-
0.119
[protein NqrC]-L-threonine
pH 9.0, temperature not specified in the publication, mutant H257t
-
additional information
additional information
ApbE activity is highly sensitive to FAD, and is saturated at 0.001 mM
-
additional information
additional information
ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed, Michaelis-Menten steady-state kinetic analysis, kinetic mechanism
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additional information
additional information
-
ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed, Michaelis-Menten steady-state kinetic analysis, kinetic mechanism
-
additional information
additional information
single-turnover kinetics
-
additional information
additional information
-
single-turnover kinetics
-
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evolution
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
evolution
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
evolution
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
evolution
ApbE is a member of a family of flavin transferases that incorporates flavin mononucleotide (FMN) to subunits of diverse respiratory complexes, which fulfill important homeostatic functions. Enzyme residue His257 is absolutely conserved in the family
evolution
FMN residue attached through a phosphoester bond is found in three types of protein architectures-prokaryotic FMN bind and NQR2 RnfD RnfE (PF03116) domains and an about 50-residue N-terminal extension of the ApbE domain in eukaryotic NADH:fumarate oxidoreductases. These three architectures are non-homologous, and their sequences have nothing in common, except for a short motif around the flavinylated residue. The motif common to all three flavinylated architectures can be depicted as Dxx(s/t)(g/s)At/s, where the last residue is the flavin acceptor. It is threonine in all characterized proteins of the first two groups, but it is sporadically replaced by serine in 3.5-5% of their putative homologues
evolution
-
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
-
evolution
-
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
-
evolution
-
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
-
evolution
-
there likely are two classes of Ftps, one associated with FAD-binding and the other with FAD hydrolysis
-
malfunction
-
enzyme inactivation results in a complete loss of the quinone reductase activity of Na+-translocating NADH:quinone oxidoreductase
malfunction
lesion in the ApbE1 gene in results in inactive Na+-translocating NADH:quinone oxidoreductase, but cytoplasmic fumarate reductase activity remains unchanged
malfunction
lesion in the ApbE2 gene in results in inactive cytoplasmic fumarate reductase, but Na+-translocating NADH:quinone oxidoreductase activity remains unchanged
malfunction
a single amino acid substitution Y60N converts it from an FAD-binding protein to a Mg2+-dependent FAD diphosphatase (Ftp_Tp-like). The engineered protein variant (Ftp_EcY60A) shows Mg2+-dependent FAD diphosphatase activity, but also retains its Mg2+-dependent FMN transferase (EC 2.7.1.180) activity on the protein substrate, indicating that the protein variant enzyme has dual activity
malfunction
a single amino acid substitution converts it from an FAD-binding protein to a Mg2+-dependent FAD diphosphatase (Ftp_Tp-like, EC 3.6.1.18)
malfunction
the replacement of the flavin acceptor threonine with alanine completely abolishes the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10-55% decrease in the flavinylation degree). Replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allows appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent
malfunction
-
enzyme inactivation results in a complete loss of the quinone reductase activity of Na+-translocating NADH:quinone oxidoreductase
-
malfunction
-
lesion in the ApbE1 gene in results in inactive Na+-translocating NADH:quinone oxidoreductase, but cytoplasmic fumarate reductase activity remains unchanged
-
malfunction
-
lesion in the ApbE2 gene in results in inactive cytoplasmic fumarate reductase, but Na+-translocating NADH:quinone oxidoreductase activity remains unchanged
-
physiological function
-
ApbE is a modifying enzyme involved in the maturation of flavoproteins. ApbE is the only protein factor required for flavinylation of subunit NqrC in Na+-translocating NADH:quinone oxidoreductase
physiological function
Na+-translocating NADH:quinone oxidoreductase maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene
physiological function
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD to a threonine residue in a target flavoprotein. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
physiological function
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD to a threonine residue in a target flavoprotein. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
physiological function
the flavin-trafficking protein (Ftp) in the syphillis spirochete Treponema pallidum (Ftp_Tp) is a bacterial metal-dependent FAD diphosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
physiological function
many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate
physiological function
substrate specificity and regulatory mechanisms, overview. Residue H257 is the residue whose deprotonation controls the activity, it plays an important role in the catalytic mechanism of ApbE. Residue His257 is indeed essential for catalysis, but not for substrate binding
physiological function
the enzyme flavinylate the redox subunit, NqrC, via its metal-dependent FMN transferase activity
physiological function
-
ApbE is a modifying enzyme involved in the maturation of flavoproteins. ApbE is the only protein factor required for flavinylation of subunit NqrC in Na+-translocating NADH:quinone oxidoreductase
-
physiological function
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD to a threonine residue in a target flavoprotein. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
-
physiological function
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD to a threonine residue in a target flavoprotein. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
-
physiological function
-
the flavin-trafficking protein (Ftp) in the syphillis spirochete Treponema pallidum (Ftp_Tp) is a bacterial metal-dependent FAD diphosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
-
physiological function
-
Na+-translocating NADH:quinone oxidoreductase maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene
-
physiological function
-
the flavin-trafficking protein (Ftp) catalyzes the transfer of the FMN moiety of FAD to a threonine residue in a target flavoprotein. Both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. Possible mechanism by which flavoproteins are generated, overview
-
additional information
the critical residue that contacts the isoalloxazine ring of FAD, is a tyrosine residue in the FAD-binding Ftps
additional information
-
the critical residue that contacts the isoalloxazine ring of FAD, is a tyrosine residue in the FAD-binding Ftps
additional information
the critical residue that contacts the isoalloxazine ring of FAD, is a tyrosine residue in the FAD-binding Ftps. FAD binding structure involving residue K207, overview
additional information
-
the critical residue that contacts the isoalloxazine ring of FAD, is a tyrosine residue in the FAD-binding Ftps. FAD binding structure involving residue K207, overview
additional information
residue His257 is located in the catalytic site and the mutational substitution does not produce major conformational changes
additional information
-
residue His257 is located in the catalytic site and the mutational substitution does not produce major conformational changes
additional information
the flavinylation motif of FRD, D-6A-5I-4S-3G-2A-1T0S+1 Q+2S+3 (zero position corresponds to Thr447 in the amino acid sequence), is mostly typical of FMN bind domain
additional information
-
the flavinylation motif of FRD, D-6A-5I-4S-3G-2A-1T0S+1 Q+2S+3 (zero position corresponds to Thr447 in the amino acid sequence), is mostly typical of FMN bind domain
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purified Ftp_Ec mutant variant Y60N complexed with ADP, hanging drop vapor diffusion method, mixing of 0.004 ml of 20 mg/ml protein in Tris, pH 7.5, and 20 mM NaCl, with 0.04 ml of reservoir solution containing 0.2 M NH4NO3, and 20% w/v PEG 3350, and 0.001 ml of 50 mM MgCl2, and 0.001 ml of 50 mM ADP, 20°C, X-ray diffraction structure determination and analysis at 1.85 A resolution, molecular replacement and modelling
purified recombinant wild-type enzyme Ftp_Ec, mutant E169K, and mutant Y60N bound to ADP, hanging drop vapor diffusion method, mixing of 0.004 ml of 20 mg/ml protein in 20 mM Tris, pH 7.5, and 20 mM NaCl, with 0.004 ml of reservoir solution containing 0.2 M NH4NO3 and 20% w/v PEG 3350 for the wild-type, and 25% w/v PEG 1500, 0.1 M MIB (malonate, imidazole and boric acid), pH 5.0 for the mutant, 20°C, X-ray diffraction structure determination and analysis at 1.75-1.88 A resolution
purified recombinant His-tagged wild-type ApbE and H257G mutant, sitting drop vapor diffusion technique, mixing of protein in 10 mM HEPES, 150 mM NaCl, 0.5 mM FAD, and 5 mM MgCl2, pH 8.0, with crystallization solution containing 0.1 M sodium acetate, 25% PEG 4000, and 8% isopropyl alcohol for the wild-type enzyme, and 0.1 M sodium acetate, 22% PEG 4000, and 0.1 M HEPES buffer, pH 7.5, for the mutant H257G enzyme, 16°C, X-ray diffraction structure determination and analysis at 1.61 A and 1.92 A resolution, respectively
analysis of X-ray crystal structure of the Vibrio cholerae Nqr complex and structures of several purified components of the complex, PDB IDs 4U9S and 4P6V
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K207A
site-directed mutagenesis, the mutant exhibits no detectable in vitro flavinylation activity
Y60A
site-directed mutagenesis, the engineered protein variant (Ftp_EcY60A) shows Mg2+-dependent FAD diphosphatase activity, but also retains its Mg2+-dependent FMN transferase (EC 2.7.1.180) activity on the protein substrate, indicating that the protein variant enzyme has dual activity
H257E
site-directed mutagenesis, almost inactive mutant
H257K
site-directed mutagenesis, almost inactive mutant
H257T
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
E169K
site-directed mutagenesis of the probabale catalytic site residue, the Ftp_EcE169K protein variant does not show binding of FAD, inactive mutant
E169K
site-directed mutagenesis, mutation of the active-site residue results in loss of FAD binding capability
Y60N
site-directed mutagenesis, a single amino acid substitution converts it from an FAD-binding protein to a Mg2+-dependent FAD diphosphatase (Ftp_Tp-like)
Y60N
site-directed mutagenesis, the single amino acid substitution converts it from an FAD-binding protein to a Mg2+-dependent FAD pdiphosphatase (Ftp_Tp-like, EC 3.6.1.18). The Ftp_EcY60A protein variant binds FAD, rapidly hydrolyzes it, and the product FMN dissociates. But the mutant also retains its Mg2+-dependent FMN transferase (EC 2.7.1.180) activity on the protein substrate. As the site of attack for the FMN transferase reaction is the beta-phosphate of the FAD, and given the large distance between the two metals in the ADP-inhibited Ftp_EcY60N structure, it is reasonable to expect that only metal site 2 requires a Mg2+ ion for this activity
H257G
site-directed mutagenesis, the mutant's flavin transfer activity is abolished. After reconstitution, the H257G shows a FAD:protein ratio nearly identical to the wild-type
H257G
site-directed mutagenesis, the turnover rates of His257 mutants are significantly smaller than those of wild-type ApbE, and mutants show increased Km values for both substrates, the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His257 mutants compared to wild-type, the mutant shows reduced activity compared to wild-type
additional information
replacement of non-Ala residues in the positions 0, -2, -3 and -6 by Ala, and Ala-1 by Val. Keeping in mind that the position +2 is occupied by a non-typical Gln in FRD, this residue is also replaced by Ala
additional information
-
replacement of non-Ala residues in the positions 0, -2, -3 and -6 by Ala, and Ala-1 by Val. Keeping in mind that the position +2 is occupied by a non-typical Gln in FRD, this residue is also replaced by Ala
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Bertsova, Y.V.; Kostyrko, V.A.; Baykov, A.A.; Bogachev, A.V.
Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na+-translocating NADH:quinone oxidoreductase
Biochim. Biophys. Acta
1837
1122-1129
2014
Klebsiella pneumoniae (B5XP00), Klebsiella pneumoniae (B5XRA9), Klebsiella pneumoniae, Klebsiella pneumoniae 204 (B5XP00), Klebsiella pneumoniae 342 (B5XRA9)
brenda
Bertsova, Y.V.; Fadeeva, M.S.; Kostyrko, V.A.; Serebryakova, M.V.; Baykov, A.A.; Bogachev, A.V.
Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins
J. Biol. Chem.
288
14276-14286
2013
Vibrio harveyi, Vibrio harveyi R3
brenda
Kostyrko, V.A.; Bertsova, Y.V.; Serebryakova, M.V.; Baykov, A.A.; Bogachev, A.V.
NqrM (DUF539) protein is required for maturation of bacterial Na+-translocating NADH:quinone oxidoreductase
J. Bacteriol.
198
655-663
2016
Vibrio harveyi (A0A3A1Q8E8), Vibrio harveyi, Vibrio harveyi ATCC 33843 (A0A3A1Q8E8)
brenda
Deka, R.K.; Brautigam, C.A.; Liu, W.Z.; Tomchick, D.R.; Norgard, M.V.
Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm
MicrobiologyOpen
5
21-38
2016
Vibrio cholerae serotype O1 (A5F5Y3), Treponema pallidum (O83774), Treponema pallidum, Escherichia coli (P0AB85), Escherichia coli, Vibrio cholerae serotype O1 Classical Ogawa 395 (A5F5Y3), Vibrio cholerae serotype O1 ATCC 39541 (A5F5Y3), Treponema pallidum Nichols (O83774), Vibrio cholerae serotype O1 O395 (A5F5Y3)
brenda
Bertsova, Y.V.; Serebryakova, M.V.; Anashkin, V.A.; Baykov, A.A.; Bogachev, A.V.
Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE
FEMS Microbiol. Lett.
366
fnz252
2019
Klebsiella pneumoniae (A0A0C7KIE8), Klebsiella pneumoniae
brenda
Fang, X.; Osipiuk, J.; Chakravarthy, S.; Yuan, M.; Menzer, W.M.; Nissen, D.; Liang, P.; Raba, D.A.; Tuz, K.; Howard, A.J.; Joachimiak, A.; Minh, D.D.L.; Juarez, O.
Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis
J. Biol. Chem.
294
13800-13810
2019
Vibrio cholerae (A0A0F4FI39), Vibrio cholerae
brenda
Fang, X.; Liang, P.; Raba, D.; Rosas-Lemus, M.; Chakravarthy, S.; Tuz, K.; Juárez, O.
Kinetic characterization of Vibrio cholerae ApbE substrate specificity and regulatory mechanisms
PLoS ONE
12
e0186805
2017
Vibrio cholerae (A0A0F4FI39)
brenda