MetaCyc Enzyme: biotin synthase

Gene: bioB Accession Numbers: EG10118 (MetaCyc), b0775, ECK0764

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of biotin synthase = [BioB]2
         biotin synthase monomer = BioB

Biotin synthase catalyzes the final reaction of biotin biosynthesis by inserting a sulfur atom between C6 and C9 of dethiobiotin in a S-adenosylmethoinine (SAM)-dependent reaction. For a long time, it was not possible to reconstitute a catalytic reaction in vitro, and uncertainty regarding the reaction mechanism, cofactor requirements, and the source of the sulfur atom remained [Jarrett05]. However, under optimal conditions with pure substrate and removal of the inhibitory 5'-deoxyadenosine product, burst kinetics and multiple turnovers of the enzyme can be observed [Farrar10].

BioB belongs to the family of "radical SAM" enzymes [Jarrett05a]. The enzyme purifies as a homodimer [Sanyal94]. It contains two distinct iron-sulfur binding sites; one carries an air-stable [2Fe-2S] cluster, and the other an air-sensitive [4Fe-4S] cluster that binds SAM and facilitates its reductive cleavage to generate a 5'-deoxyadenosyl radical (dA·) which activates dethiobiotin [Ollagnierde02, Ugulava02, Cosper02, Cosper02a, Benda02, Ugulava03, Berkovitch04, Jameson04]. Recent experiments have suggested that the [2Fe-2S] cluster is the source of the sulfur atom. Consistent with its proposed role as the sulfur donor, degradation of the [2Fe-2S] cluster [Jameson04] as well as exchange of sulfur atoms between the [2Fe-2S] and [4Fe-4S] clusters [Tse06] is observed during turnover of the enzyme. An enzyme containing a [2Fe-2Se] cluster can produce selenobiotin in vitro [Tse06]. The enzyme does not appear to be able to function catalytically in vitro [Tse04]; BioB is catalytically active in vivo, but it is thought that reconstitution of the [2Fe-2S] cluster makes the enzyme susceptible to proteolytic degradation [ChoiRhee05]. Studies of BioB holoenzyme and two cluster-deficient forms show that loss of the [2Fe-2S] cluster does not significantly destabilize the BioB dimer. Loss of both Fe-S clusters decreases the thermal stability of BioB and may lead to unfolding near specific Arg residues. In vivo, BioB is more rapidly degraded under iron-limited growth conditions. These results support a model of balanced Fe-S cluster repair and protein degradation [Reyda08].

Reassembly of the [2Fe-2S] cluster involves the chaperone HscA, which interacts with the apo form of biotin synthase and improves the efficiency of assembly of the [2Fe-2S] cluster. The HscA-BioB complex also binds the Fe-S cluster scaffold protein IscU [Reyda09].

The enzyme forms 9-mercaptodethiobiotin as an intermediate that remains tightly bound [Taylor08a].

A crystal structure of biotin synthase has been solved at 3.4 Å resolution [Berkovitch04]. Reaction mechanisms involving either the [2Fe-2S] cluster [Bui98, Ugulava01] or the cofactor PLP [OllagnierDeChou02, OllagnierdeChou02] were proposed; however, the crystal structure of BioB [Berkovitch04] and the cofactor composition of the enzyme [Cosper04] do not support involvement of PLP. Finally, genetic experiments using a pdxH mutant unable to produce PLP showed that BioB activity does not depend on PLP in vivo [AbdelHamid07a].

Site-directed mutagenesis of conserved cysteine residues identified the sites involved in binding the iron-sulfur clusters [Hewitson00, Farh01, Hewitson02] as well as active site residues [Lotierzo06, Lotierzo09]. The conserved N153 and D155 residues appear to play a role in retaining intermediates during the catalytic cycle [Farrar09]. Mutagenesis of the atypical guanidinium metal ligand Arg260 showed that this residue is not essential for catalysis [Broach06].

Reviews: [Jarrett05a, Lotierzo05, Booker07]

Map Position: [808,567 -> 809,607]

Molecular Weight of Polypeptide: 38.648 kD (from nucleotide sequence)

Molecular Weight of Multimer: 82 kD (experimental) [Sanyal94]

pI: 5.55

Unification Links: ASAP:ABE-0002644 , CGSC:958 , DIP:DIP-9220N , EchoBASE:EB0116 , EcoGene:EG10118 , EcoliWiki:b0775 , Mint:MINT-1292998 , ModBase:P12996 , OU-Microarray:b0775 , PortEco:bioB , PR:PRO_000022222 , Pride:P12996 , Protein Model Portal:P12996 , RefSeq:NP_415296 , RegulonDB:EG10118 , SMR:P12996 , String:511145.b0775 , UniProt:P12996

Relationship Links: InterPro:IN-FAMILY:IPR002684 , InterPro:IN-FAMILY:IPR006638 , InterPro:IN-FAMILY:IPR007197 , InterPro:IN-FAMILY:IPR010722 , InterPro:IN-FAMILY:IPR013785 , InterPro:IN-FAMILY:IPR024177 , PDB:Structure:1R30 , Pfam:IN-FAMILY:PF04055 , Pfam:IN-FAMILY:PF06968 , Smart:IN-FAMILY:SM00729 , Smart:IN-FAMILY:SM00876

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0009102 - biotin biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA06, GOA01a, Rolfe70]
Molecular Function: GO:0004076 - biotin synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Lotierzo06]
GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Reyda09]
GO:0051537 - 2 iron, 2 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, Cosper04]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, Cosper04]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0005506 - iron ion binding Inferred by computational analysis [GOA06]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11, GOA06, GOA01a]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers biotin

Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

Enzymatic reaction of: biotin synthase

Synonyms: dethiobiotin:sulfur sulfurtransferase

EC Number:

a sulfurated [sulfur carrier] + dethiobiotin + 2 S-adenosyl-L-methionine + 2 a reduced [2Fe-2S] ferredoxin <=> an unsulfurated [sulfur carrier] + biotin + 2 5'-deoxyadenosine + 2 L-methionine + 2 an oxidized [2Fe-2S] ferredoxin

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for dethiobiotin: 9-mercaptodethiobiotin [Lotierzo05 ]

In Pathways: biotin biosynthesis I , biotin biosynthesis from 8-amino-7-oxononanoate I

Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

While both 5'-deoxyadenosine and L-methionine inhibit the enzyme moderately, a combination of both in equimolar concentrations cooperatively inhibit biotin synthase. The in vivo concentration of 5'-deoxyadenosine is likely sufficiently low that this inhibition is not physiologically relevant [Farrar10].

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Cosper04], a [2Fe-2S] iron-sulfur cluster [Cosper04]

Inhibitors (Unknown Mechanism): sinefungin [Farrar10] , L-methionine [Farrar10] , S-adenosyl-L-homocysteine [Farrar10] , 5'-deoxyadenosine [ChoiRhee05a, Farrar10, Challand09]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
[Fugate12, BRENDA14]
[Taylor08a, BRENDA14]
[Sanyal94, BRENDA14]
[Kiyasu02, BRENDA14]
[Farh01, BRENDA14]
[Farrar10, BRENDA14]

T(opt): 37 °C [BRENDA14, Farrar09]

pH(opt): 8 [BRENDA14, Farrar09]

Sequence Features

Feature Class Location Citations Comment
Mutagenesis-Variant 53
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 53
UniProt: Iron-sulfur 1 (4Fe-4S-S-AdoMet).
Mutagenesis-Variant 57
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 57
UniProt: Iron-sulfur 1 (4Fe-4S-S-AdoMet).
Mutagenesis-Variant 60
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 60
UniProt: Iron-sulfur 1 (4Fe-4S-S-AdoMet).
Sequence-Conflict 63
[Otsuka88, UniProt10a]
UniProt: (in Ref. 1; AAA23515);
Mutagenesis-Variant 97
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 97
UniProt: Iron-sulfur 2 (2Fe-2S).
Mutagenesis-Variant 128
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 128
UniProt: Iron-sulfur 2 (2Fe-2S).
Mutagenesis-Variant 151
[Lotierzo06, UniProt15]
UniProt: Total loss of activity.
Mutagenesis-Variant 152
[Lotierzo06, UniProt15]
UniProt: Weak activity.
Mutagenesis-Variant 153
[Lotierzo06, UniProt15]
UniProt: Total loss of activity.
Mutagenesis-Variant 155
[Lotierzo06, UniProt15]
UniProt: Total loss of activity.
Mutagenesis-Variant 188
[Hewitson02, Ollagnierde02, UniProt15]
UniProt: Total loss of activity.
Metal-Binding-Site 188
UniProt: Iron-sulfur 2 (2Fe-2S).
Metal-Binding-Site 260
UniProt: Iron-sulfur 2 (2Fe-2S).

1/26/1998 (pkarp) Merged genes G8004/bioB and EG10118/bioB


AbdelHamid07a: Abdel-Hamid AM, Cronan JE (2007). "In vivo resolution of conflicting in vitro results: synthesis of biotin from dethiobiotin does not require pyridoxal phosphate." Chem Biol 14(11);1215-20. PMID: 18022560

Benda02: Benda R, Tse Sum Bui B, Schunemann V, Florentin D, Marquet A, Trautwein AX (2002). "Iron-sulfur clusters of biotin synthase in vivo: a Mossbauer study." Biochemistry 41(50);15000-6. PMID: 12475249

Berkovitch04: Berkovitch F, Nicolet Y, Wan JT, Jarrett JT, Drennan CL (2004). "Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme." Science 303(5654);76-9. PMID: 14704425

Booker07: Booker SJ, Cicchillo RM, Grove TL (2007). "Self-sacrifice in radical S-adenosylmethionine proteins." Curr Opin Chem Biol 11(5);543-52. PMID: 17936058

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014."

Broach06: Broach RB, Jarrett JT (2006). "Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260." Biochemistry 45(47);14166-74. PMID: 17115711

Bui98: Bui BT, Florentin D, Fournier F, Ploux O, Mejean A, Marquet A (1998). "Biotin synthase mechanism: on the origin of sulphur." FEBS Lett 1998;440(1-2);226-30. PMID: 9862460

Challand09: Challand MR, Ziegert T, Douglas P, Wood RJ, Kriek M, Shaw NM, Roach PL (2009). "Product inhibition in the radical S-adenosylmethionine family." FEBS Lett 583(8);1358-62. PMID: 19328201

ChoiRhee05: Choi-Rhee E, Cronan JE (2005). "Biotin synthase is catalytic in vivo, but catalysis engenders destruction of the protein." Chem Biol 12(4);461-8. PMID: 15850983

ChoiRhee05a: Choi-Rhee E, Cronan JE (2005). "A nucleosidase required for in vivo function of the S-adenosyl-L-methionine radical enzyme, biotin synthase." Chem Biol 12(5);589-93. PMID: 15911379

Cosper02: Cosper MM, Jameson GN, Eidsness MK, Huynh BH, Johnson MK (2002). "Recombinant Escherichia coli biotin synthase is a [2Fe-2S](2+) protein in whole cells." FEBS Lett 529(2-3);332-6. PMID: 12372623

Cosper02a: Cosper MM, Jameson GN, Davydov R, Eidsness MK, Hoffman BM, Huynh BH, Johnson MK (2002). "The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine." J Am Chem Soc 124(47);14006-7. PMID: 12440894

Cosper04: Cosper MM, Jameson GN, Hernandez HL, Krebs C, Huynh BH, Johnson MK (2004). "Characterization of the cofactor composition of Escherichia coli biotin synthase." Biochemistry 43(7);2007-21. PMID: 14967041

Farh01: Farh L, Hwang SY, Steinrauf L, Chiang HJ, Shiuan D (2001). "Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach." J Biochem (Tokyo) 130(5);627-35. PMID: 11686925

Farrar09: Farrar CE, Jarrett JT (2009). "Protein residues that control the reaction trajectory in S-adenosylmethionine radical enzymes: mutagenesis of asparagine 153 and aspartate 155 in Escherichia coli biotin synthase." Biochemistry 48(11);2448-58. PMID: 19199517

Farrar10: Farrar CE, Siu KK, Howell PL, Jarrett JT (2010). "Biotin synthase exhibits burst kinetics and multiple turnovers in the absence of inhibition by products and product-related biomolecules." Biochemistry 49(46);9985-96. PMID: 20961145

Fugate12: Fugate CJ, Jarrett JT (2012). "Biotin synthase: insights into radical-mediated carbon-sulfur bond formation." Biochim Biophys Acta 1824(11);1213-22. PMID: 22326745

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Hewitson00: Hewitson KS, Baldwin JE, Shaw NM, Roach PL (2000). "Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase." FEBS Lett 466(2-3);372-6. PMID: 10682863

Hewitson02: Hewitson KS, Ollagnier-de Choudens S, Sanakis Y, Shaw NM, Baldwin JE, Munck E, Roach PL, Fontecave M (2002). "The iron-sulfur center of biotin synthase: site-directed mutants." J Biol Inorg Chem 7(1-2);83-93. PMID: 11862544

Jameson04: Jameson GN, Cosper MM, Hernandez HL, Johnson MK, Huynh BH (2004). "Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase." Biochemistry 43(7);2022-31. PMID: 14967042

Jarrett05: Jarrett JT (2005). "Biotin synthase: enzyme or reactant?." Chem Biol 12(4);409-10. PMID: 15850974

Jarrett05a: Jarrett JT (2005). "The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase." Arch Biochem Biophys 433(1);312-21. PMID: 15581586

Kiyasu02: Kiyasu T, Asakura A, Nagahashi Y, Hoshino T (2002). "Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems." Arch Microbiol 179(1);26-32. PMID: 12471501

Lotierzo05: Lotierzo M, Tse Sum Bui B, Florentin D, Escalettes F, Marquet A (2005). "Biotin synthase mechanism: an overview." Biochem Soc Trans 33(Pt 4);820-3. PMID: 16042606

Lotierzo06: Lotierzo M, Raux E, Tse Sum Bui B, Goasdoue N, Libot F, Florentin D, Warren MJ, Marquet A (2006). "Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif." Biochemistry 45(40);12274-81. PMID: 17014080

Lotierzo09: Lotierzo M, Bui BT, Leech HK, Warren MJ, Marquet A, Rigby SE (2009). "Iron-sulfur cluster dynamics in biotin synthase: a new [2Fe-2S](1+) cluster." Biochem Biophys Res Commun 381(4);487-90. PMID: 19245793

Ollagnierde02: Ollagnier-de Choudens S, Sanakis Y, Hewitson KS, Roach P, Munck E, Fontecave M (2002). "Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli." J Biol Chem 277(16);13449-54. PMID: 11834738

OllagnierdeChou02: Ollagnier-de-Choudens S, Mulliez E, Fontecave M (2002). "The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies." FEBS Lett 532(3);465-8. PMID: 12482614

OllagnierDeChou02: Ollagnier-De-Choudens S, Mulliez E, Hewitson KS, Fontecave M (2002). "Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase." Biochemistry 41(29);9145-52. PMID: 12119030

Otsuka88: Otsuka AJ, Buoncristiani MR, Howard PK, Flamm J, Johnson C, Yamamoto R, Uchida K, Cook C, Ruppert J, Matsuzaki J (1988). "The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon." J Biol Chem 263(36);19577-85. PMID: 3058702

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Reyda08: Reyda MR, Dippold R, Dotson ME, Jarrett JT (2008). "Loss of iron-sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation." Arch Biochem Biophys 471(1):32-41. PMID: 18155152

Reyda09: Reyda MR, Fugate CJ, Jarrett JT (2009). "A complex between biotin synthase and the iron-sulfur cluster assembly chaperone HscA that enhances in vivo cluster assembly." Biochemistry 48(45);10782-92. PMID: 19821612

Rolfe70: Rolfe B (1970). "Lambda phage transduction of the bio A locus of Escherichia coli." Virology 42(3);643-61. PMID: 4921685

Sanyal94: Sanyal I, Cohen G, Flint DH (1994). "Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product." Biochemistry 1994;33(12);3625-31. PMID: 8142361

Taylor08a: Taylor AM, Farrar CE, Jarrett JT (2008). "9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase." Biochemistry 47(35);9309-17. PMID: 18690713

Tse04: Tse Sum Bui B, Lotierzo M, Escalettes F, Florentin D, Marquet A (2004). "Further investigation on the turnover of Escherichia coli biotin synthase with dethiobiotin and 9-mercaptodethiobiotin as substrates." Biochemistry 43(51);16432-41. PMID: 15610037

Tse06: Tse Sum Bui B, Mattioli TA, Florentin D, Bolbach G, Marquet A (2006). "Escherichia coli biotin synthase produces selenobiotin. Further evidence of the involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step." Biochemistry 45(11);3824-34. PMID: 16533066

Ugulava01: Ugulava NB, Sacanell CJ, Jarrett JT (2001). "Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe-2S] cluster accompanies sulfur insertion." Biochemistry 40(28);8352-8. PMID: 11444982

Ugulava02: Ugulava NB, Surerus KK, Jarrett JT (2002). "Evidence from Mossbauer spectroscopy for distinct [2Fe-2S](2+) and [4Fe-4S](2+) cluster binding sites in biotin synthase from Escherichia coli." J Am Chem Soc 124(31);9050-1. PMID: 12148999

Ugulava03: Ugulava NB, Frederick KK, Jarrett JT (2003). "Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB." Biochemistry 42(9);2708-19. PMID: 12614166

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 19.0 on Tue Oct 13, 2015, BIOCYC14A.