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Information on EC 1.13.11.79 - aerobic 5,6-dimethylbenzimidazole synthase
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1.13.11.79 aerobic 5,6-dimethylbenzimidazole synthaseIUBMB Comments
The enzyme catalyses a complex oxygen-dependent conversion of reduced flavin mononucleotide to form 5,6-dimethylbenzimidazole, the lower ligand of vitamin B12. This conversion involves many sequential steps in two distinct stages, and an alloxan intermediate that acts as a proton donor, a proton acceptor, and a hydride acceptor . The C-2 of 5,6-dimethylbenzimidazole is derived from C-1' of the ribityl group of FMNH2 and 2-H from the ribityl 1'-pro-S hydrogen. While D-erythrose 4-phosphate has been shown to be one of the byproducts, the nature of the other product(s) has not been verified yet.
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The expected taxonomic range for this enzyme is: Bacteria, Archaea
Reaction Schemes
+
=
+
+
other product(s)
Synonyms
blub/cobt2, dmb-synthase, flavin destructase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
BluB
bluB/cobT2
DMB-synthase
EC 1.14.99.40
-
-
formerly
-
flavin destructase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
FMNH2 + O2 = 5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + other product(s)
-
-
-
-
FMNH2 + O2 = 5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + other product(s)
intermediate-assisted reaction mechanism in multifunctional catalysis, conversion of FMNH2 to 5,6-dimethylbenzimidazole proceeds via more than 14 steps in two distinct stages, modeling, analysis of crystal structure and geometry optimization, detailed overview
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SYSTEMATIC NAME
IUBMB Comments
FMNH2 oxidoreductase (5,6-dimethylbenzimidazole forming)
The enzyme catalyses a complex oxygen-dependent conversion of reduced flavin mononucleotide to form 5,6-dimethylbenzimidazole, the lower ligand of vitamin B12. This conversion involves many sequential steps in two distinct stages, and an alloxan intermediate that acts as a proton donor, a proton acceptor, and a hydride acceptor [4]. The C-2 of 5,6-dimethylbenzimidazole is derived from C-1' of the ribityl group of FMNH2 and 2-H from the ribityl 1'-pro-S hydrogen. While D-erythrose 4-phosphate has been shown to be one of the byproducts, the nature of the other product(s) has not been verified yet.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
FMNH2 + NADH + H+ + O2 + alloxan
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + dialuric acid
FMNH2 + NADH + H+ + O2 + H2O
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + ?
-
-
-
?
FMNH2 + NADPH + H+ + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NADP+ + ?
-
-
-
-
r
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + dialiric acid + pyrimidinetrione + alloxan
-
the rate of formation of the intermediate product unprotonated C4a-peroxyflavin is influenced by substitutions at C8 of FMN
-
-
?
FMNH2 + NADH + H+ + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + ?
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-
-
-
?
FMNH2 + NADH + H+ + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + ?
-
5,6-dimethylbenzimidazole is the lower axial ligand of vitamin B12, i.e. cobalamin
-
?
FMNH2 + NADH + H+ + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + ?
BluB fragments a flavin isoalloxazine ring
-
-
?
FMNH2 + NADH + H+ + O2 + alloxan
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + dialuric acid
-
-
-
-
?
FMNH2 + NADH + H+ + O2 + alloxan
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + dialuric acid
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in the first stage, BluB catalyzes the incorporation of dioxygen, and the fragmentation of the isoalloxazine ring of FMNH2 to form alloxan and the ribityl dimethylphenylenediimine. In the second stage, BluB exploits alloxan as a multifunctional cofactor, such as a proton donor, a proton acceptor, and a hydride acceptor, to catalyze the remaining no fewer than 10 steps of the reaction. The retro-aldol cleavage of the C1'-C2' bond of intermediate ribityl dimethylphenylenediimine is the rate-determining step, active site model of BluB, overview. The highly conserved residue Asp32 plays critical roles in multiple steps of the conversion by serving as a proton acceptor or a proton shuttle, and another conserved residue Ser167 plays its catalytic role mainly in the rate-determining step by stabilizing the protonated retro-aldol precursor. Formation of peroxyflavin intermediate, overview
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + ?
-
FMNH2 reacts with oxygen to yield flavin hydroperoxide, which then undergoes a ring expansion with loss of a water molecule to produce intermediate A. This disintegrates further as a result of four hydrolysis reactions in which it is attacked by water molecules. The resulting diaminobenzene product undergoes two oxidation reactions, to form 5,6-dimethylbenzimidazole
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + other product(s)
-
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + other product(s)
-
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + urea + ?
-
-
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + urea + ?
-
-
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
FMNH2 + NADH + H+ + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + ?
-
5,6-dimethylbenzimidazole is the lower axial ligand of vitamin B12, i.e. cobalamin
-
?
FMNH2 + NADH + H+ + O2 + alloxan
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + NAD+ + dialuric acid
-
-
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + dialiric acid + pyrimidinetrione + alloxan
-
the rate of formation of the intermediate product unprotonated C4a-peroxyflavin is influenced by substitutions at C8 of FMN
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + ?
-
FMNH2 reacts with oxygen to yield flavin hydroperoxide, which then undergoes a ring expansion with loss of a water molecule to produce intermediate A. This disintegrates further as a result of four hydrolysis reactions in which it is attacked by water molecules. The resulting diaminobenzene product undergoes two oxidation reactions, to form 5,6-dimethylbenzimidazole
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + urea + ?
-
-
-
-
?
FMNH2 + O2
5,6-dimethylbenzimidazole + D-erythrose 4-phosphate + urea + ?
-
-
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
FMN/FMNH2 dissociation constants and free energy of binding of wild-type and mutant enzymes, overview
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additional information
additional information
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FMN/FMNH2 dissociation constants and free energy of binding of wild-type and mutant enzymes, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
physiological function
additional information
metabolism
BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of Propionibacterium freudenreichii
metabolism
-
the enzyme gene is required for aerobic 5,6-dimethylbenzimidazole synthesis for cobalamin production
metabolism
-
BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of Propionibacterium freudenreichii
-
physiological function
-
a Tn5 insertion mutant is unable to grow in minimal media and fails to establish a symbiosis with alfalfa. Effect can be rescued by addition of vitamin B12 or of 5,6-dimethylbenzimidazole. Mutant does not produce cobalamin unless 5,6-dimethylbenzimidazole is supplied
physiological function
enzyme deletion mutant is unable to convert Mg-protoporphyrin IX monomethyl ester into protochlorophyllide, mutant starin grows poorly under anoxic photoheterotrophic conditions
physiological function
the enzyme is involved in production of vitamin B12, a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Fusion enzyme BluB/CobT2 is efficient in metabolite channeling, and the enzymes' inability to react with adenine, a lower ligand present in the pseudovitamin, reveals a mechanism favoring the production of the active form of the vitamin, requirement of oxygen for DMBI synthesis, since the organism does not synthesize cobalamin. BluB is responsible for the formation of DMBI from FMNH2 in the presence of oxygen, BluB/CobT2 activates DMBI into alpha-RP in the presence of NaMN
physiological function
-
the enzyme is involved in production of vitamin B12, a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Fusion enzyme BluB/CobT2 is efficient in metabolite channeling, and the enzymes' inability to react with adenine, a lower ligand present in the pseudovitamin, reveals a mechanism favoring the production of the active form of the vitamin, requirement of oxygen for DMBI synthesis, since the organism does not synthesize cobalamin. BluB is responsible for the formation of DMBI from FMNH2 in the presence of oxygen, BluB/CobT2 activates DMBI into alpha-RP in the presence of NaMN
-
additional information
identification of catalytic residues involved in the reaction, mutational analysis, overview. The enzyme shows interactions with the phosphate group and ribityl tail of FMN
additional information
-
identification of catalytic residues involved in the reaction, mutational analysis, overview. The enzyme shows interactions with the phosphate group and ribityl tail of FMN
Show AA Sequence and Transmembrane Helices (2849 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D32N
D32N/S167G
site-directed mutagenesis, the mutant shows no activity
G110S
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
G133S
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
G61D
M140I
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
M94I
P202L
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
R30C
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
R30H
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
S167G
D32N
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
D32N
-
the mutant is specifically impaired in the flavin destruction phase of the reaction of the wild type enzyme
G61D
site-directed mutagenesis, the mutant shows no 5,6-dimethylbenzimidazole forming activity
M94I
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
S167G
site-directed mutagenesis, the mutant shows higly reduced activity compared to the wild-type enzyme
S167G
-
the mutant is specifically impaired in the flavin destruction phase of the reaction of the wild type enzyme
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
metal affinity chromatography
recombinant HaloTag-tagged enzyme from Escherichia coli strain KRX
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Salmonella enterica strain JE9385 and in Escherichia coli
gene bluB/cobT2, a natural fusion gene coding for a predicted phosphoribosyltransferase/nitroreductase, sequence comparisons, recombinant expression of HaloTag-tagged enzyme in Escherichia coli strain KRX
expressed in Salmonella enterica strain JE9385 and in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gray, M.J.; Escalante-Semerena, J.C.
Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12
Proc. Natl. Acad. Sci. USA
104
2921-2926
2007
Rhodospirillum rubrum (Q2RNG5)
brenda
Taga, M.; Larsen, N.; Howard-Jones, A.; Walsh, C.; Walker, G.
BluB cannibalizes flavin to form the lower ligand of vitamin B12
Nature
446
449-453
2007
Sinorhizobium meliloti
brenda
Campbell, G.; Taga, M.; Mistry, K.; Lloret, J.; Anderson, P.; Roth, J.; Walker, G.
Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12
Proc. Natl. Acad. Sci. USA
103
4634-4639
2006
Sinorhizobium meliloti
brenda
Wang, X.L.; Quan, J.M.
Intermediate-assisted multifunctional catalysis in the conversion of flavin to 5,6-dimethylbenzimidazole by BluB: a density functional theory study
J. Am. Chem. Soc.
133
4079-4091
2011
Sinorhizobium meliloti
brenda
Collins, H.F.; Biedendieck, R.; Leech, H.K.; Gray, M.; Escalante-Semerena, J.C.; McLean, K.J.; Munro, A.W.; Rigby, S.E.; Warren, M.J.; Lawrence, A.D.
Bacillus megaterium has both a functional BluB protein required for DMB synthesis and a related flavoprotein that forms a stable radical species
PLoS ONE
8
e55708
2013
Priestia megaterium, Rhodobacter capsulatus
brenda
Yu, T.Y.; Mok, K.C.; Kennedy, K.J.; Valton, J.; Anderson, K.S.; Walker, G.C.; Taga, M.E.
Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB
Protein Sci.
21
839-849
2012
Sinorhizobium meliloti (Q92PC8), Sinorhizobium meliloti
brenda
Ealick, S.; Begley, T.
Biochemistry: Molecular cannibalism
Nature
446
387-389
2007
Sinorhizobium meliloti
brenda
Deptula, P.; Kylli, P.; Chamlagain, B.; Holm, L.; Kostiainen, R.; Piironen, V.; Savijoki, K.; Varmanen, P.
BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B12 by Propionibacterium freudenreichii
Microb. Cell Fact.
14
186
2015
Propionibacterium freudenreichii subsp. shermanii (D7GJ95), Propionibacterium freudenreichii subsp. shermanii DSM 4902 (D7GJ95)
brenda
Hazra, A.; Ballou, D.; Taga, M.
Unique biochemical and sequence features enable BluB to destroy flavin and distinguish BluB from the flavin monooxygenase superfamily
Biochemistry
57
1748-1757
2018
Sinorhizobium meliloti
brenda
Walworth, N.; Lee, M.; Suffridge, C.; Qu, P.; Fu, F.; Saito, M.; Webb, E.; Sanudo-Wilhelmy, S.; Hutchins, D.
Functional genomics and phylogenetic evidence suggest genus-wide cobalamin production by the globally distributed marine nitrogen fixer Trichodesmium
Front. Microbiol.
9
189
2018
Trichodesmium sp.
brenda
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