Biotin—(acetyl-CoA-carboxylase) ligase

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Biotin—[acetyl-CoA-carboxylase] ligase
Biotin—[acetyl-CoA-carboxylase] ligase
Identifiers
EC no.	6.3.4.15
CAS no.	37340-95-7
Databases
BRENDA	enzyme data
ExPASy	NiceZyme view
KEGG	enzyme entry
MetaCyc	metabolic pathway
Rhea	reactions
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / QuickGO
PMC
Search
PMC	articles
PubMed	articles
NCBI	proteins

Biotin—(acetyl-CoA-carboxylase) ligase, more commonly known as BirA, is a 35kD enzyme found in prokaryotes, most notably Escherichia coli. It plays a central role in the metabolism of biotin (also known as vitamin B7) by performing two distinct functions: it acts as a biotin protein ligase (EC 6.3.4.15), catalyzing the covalent attachment of biotin to its target proteins, and as a transcriptional repressor, controlling the expression of the biotin biosynthesis (bio-)operon.^[1]

Due to the high specificity of its ligase activity and the exceptional strength of the resulting biotin-avidin interaction - the binding of biotin and avidin is among the strongest noncovalent interactions known^[2]^[3] -, BirA has been extensively repurposed as a powerful tool in molecular biology, proteomics, and biotechnology. Engineered variants of BirA are foundational to techniques for site-specific protein labeling and proximity-dependent identification of protein interaction networks.^[4]

Nomenclature and Classification

The systematic name of this enzyme class is biotin:apo-[acetyl-CoA:carbon-dioxide ligase (ADP-forming)] ligase (AMP-forming). Other names in common use include:^[5]

birA (gene name)
HLCS (gene name)
HCS1 (gene name)
biotin-[acetyl-CoA carboxylase] synthetase
biotin-[acetyl coenzyme A carboxylase] synthetase
acetyl coenzyme A holocarboxylase synthetase
acetyl CoA holocarboxylase synthetase
Biotin holoenzyme synthetase
biotin:apocarboxylase ligase
biotin—[acetyl-CoA-carboxylase] ligase

It belongs to the family of ligases, specifically those forming carbon-nitrogen bonds as acid-D-amino-acid ligases (peptide synthases).

Biological Function in E. coli

In its native host, BirA acts as a homeostatic regulator of biotin. BirA's primary catalytic function is to attach a molecule of D-biotin to a specific lysine residue on an acceptor protein. This post-translational modification is essential for the function of biotin-dependent carboxylases. In E. coli, the sole natural substrate for BirA is the Biotin Carboxyl Carrier Protein (BCCP), a subunit of the enzyme Acetyl-CoA Carboxylase (ACC).^[6]

The biotinylated ACC is critical for the first step of fatty acid synthesis: the carboxylation of acetyl-CoA to produce malonyl-CoA. The reaction proceeds as follows:

Biotin + Apo-BCCP + ATP $\rightleftharpoons$ Holo-BCCP + AMP + PP_i

Without a functional BirA, BCCP remains in its apo- (unbiotinylated) form, rendering ACC inactive and halting fatty acid synthesis, which is lethal to the cell. BirA also functions as a DNA-binding protein that represses the transcription of the bio-operon (bioABCDE), which contains the genes for the biotin synthesis pathway. This regulatory function is allosterically controlled by the concentration of the catalytic intermediate, biotinyl-5'-AMP.^[7]

When biotin is abundant, BirA synthesizes biotinyl-5'-AMP. This intermediate binds tightly within the BirA active site, inducing a major conformational change. In this "holo" state, the BirA dimer binds with high affinity to the bio-operator (bioO) DNA sequence, physically blocking RNA polymerase and shutting down transcription of the bio operon.
When biotin is scarce, no biotinyl-5'-AMP is formed. BirA remains in its "apo" conformation, which has a very low affinity for the bioO sequence. The operator site remains unoccupied, allowing for the transcription of the bio-operon and the synthesis of more biotin.

Structure and Mechanism

The E. coli BirA protein is a homodimer, with each monomer having a molecular weight of approximately 35.4 kDa. Each monomer is composed of three distinct domains:

N-terminal Domain: Contains a classic helix-turn-helix (HTH) DNA-binding motif. In the apo-enzyme, this domain is highly flexible and disordered.
Central Catalytic Domain: The largest domain, which forms the active site. It contains the binding pockets for ATP and biotin and is responsible for both steps of the ligase reaction.
C-terminal Domain: Contributes to the dimerization interface.

The transition from the ligase-competent to the repressor-competent state is driven by the binding of biotinyl-5'-AMP, which orders a flexible loop in the central domain. This change is allosterically transmitted to the N-terminal domain, causing it to lock into a fixed orientation that is optimal for dimerization and high-affinity DNA binding. The biotinylation reaction occurs in two discrete steps within the same active site:^[8]

BirA uses ATP to activate the carboxyl group of biotin, forming a high-energy mixed anhydride intermediate, biotinyl-5'-adenylate (biotinyl-5'-AMP), and releasing pyrophosphate (PP_i).

Biotin + ATP ⇌ Biotinyl-5'-AMP + PP_i

The activated biotinyl group is transferred from AMP to the ε-amino group of the specific target lysine residue on the acceptor protein (e.g., BCCP). This forms a stable amide bond.

Biotinyl-5'-AMP + Apo-protein → Biotinylated-protein + AMP

Applications in Biotechnology

Site-Specific Biotinylation (AviTag)

The natural recognition sequence for BirA on BCCP has been minimized to a 15-amino-acid peptide, commonly known as the AviTag (sequence: GLNDIFEAQKIEWHE).^[9] By genetically fusing the AviTag to a protein of interest (POI), researchers can use BirA to specifically biotinylate that protein at a single, known site.

References

↑ Chapman-Smith, A.; Mulhern, T. D.; Whelan, F.; Cronan, J. E.; Wallace, J. C. (2001-12-01). "The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity". Protein Science. 10 (12): 2608–2617. doi:10.1110/ps.22401. ISSN 0961-8368. PMC 2374043. PMID 11714929.
↑ "Dissociation constant of avidin and biotin - Generic - BNID 107216". bionumbers.hms.harvard.edu. Retrieved 2025-07-07.
↑ "Dissociation constant of the streptavidin and - Generic - BNID 114157". bionumbers.hms.harvard.edu. Retrieved 2025-07-07.
↑ Roux, Kyle J.; Kim, Dae In; Raida, Manfred; Burke, Brian (2012-03-19). "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells". The Journal of Cell Biology. 196 (6): 801–810. doi:10.1083/jcb.201112098. ISSN 1540-8140. PMC 3308701. PMID 22412018.
↑ "ExplorEnz: EC 6.3.4.15". www.enzyme-database.org. Retrieved 2025-07-07.
↑ Cronan, J. E. (1989-08-11). "The E. coli bio operon: transcriptional repression by an essential protein modification enzyme". Cell. 58 (3): 427–429. doi:10.1016/0092-8674(89)90421-2. ISSN 0092-8674. PMID 2667763.
↑ Wilson, K. P.; Shewchuk, L. M.; Brennan, R. G.; Otsuka, A. J.; Matthews, B. W. (1992-10-01). "Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains". Proceedings of the National Academy of Sciences of the United States of America. 89 (19): 9257–9261. Bibcode:1992PNAS...89.9257W. doi:10.1073/pnas.89.19.9257. ISSN 0027-8424. PMC 50105. PMID 1409631.
↑ Beckett, Dorothy (2009-01-01). "Biotin sensing at the molecular level". The Journal of Nutrition. 139 (1): 167–170. doi:10.3945/jn.108.095760. ISSN 1541-6100. PMC 2646212. PMID 19056812.
↑ Schatz, P. J. (1993-10-01). "Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli". Bio/Technology. 11 (10): 1138–1143. doi:10.1038/nbt1093-1138. ISSN 0733-222X. PMID 7764094.

v t e Enzymes: CO CS and CN ligases (EC 6.1-6.3)
6.1: Carbon-Oxygen	Aminoacyl tRNA synthetase Alanine Arginine Asparagine Aspartate Cysteine D-Alanine—poly(phosphoribitol) ligase Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
6.2: Carbon-Sulfur	(2,2,3-trimethyl-5-oxocyclopent-3-enyl)acetyl-CoA synthase 2-furoate—CoA ligase 3-alpha,7-alpha-dihydroxy-5-beta-cholestanate—CoA ligase 3-hydroxybenzoate—CoA ligase 3-hydroxypropionyl-CoA synthase 4-chlorobenzoate—CoA ligase 4-Coumarate-CoA ligase 4-hydroxybenzoate—CoA ligase 6-carboxyhexanoate—CoA ligase Acetate—ACP ligase Acetate—CoA ligase (ADP-forming) Acetoacetate—CoA ligase Acetyl—CoA synthetase Acid—CoA ligase (GDP-forming) Anthranilate—CoA ligase Arachidonate—CoA ligase Benzoate—CoA ligase Biotin—CoA ligase (butirosin acyl-carrier protein)—L-glutamate ligase Butyrate—CoA ligase Cholate—CoA ligase Citrate—CoA ligase (citrate (pro-3S)-lyase) ligase Dicarboxylate—CoA ligase Glutarate—CoA ligase Long-chain-fatty-acid—ACP ligase Long-chain-fatty-acid—CoA ligase Malate—CoA ligase Malonate—CoA ligase o-Succinylbenzoate—CoA ligase Oxalate—CoA ligase Phenylacetate—CoA ligase Phytanate—CoA ligase Propionate—CoA ligase Succinate—CoA ligase (ADP-forming) Succinate—CoA ligase (GDP-forming) Succinyl—CoA synthetase trans-Feruloyl—CoA synthase
6.3: Carbon-Nitrogen	Glutamine synthetase Ubiquitin ligase Cullin Von Hippel–Lindau tumor suppressor UBE3A Mdm2 Anaphase-promoting complex UBR1 Glutathione synthetase CTP synthetase Adenylosuccinate synthase Argininosuccinate synthase Holocarboxylase synthetase GMP synthase Asparagine synthetase Carbamoyl phosphate synthetase I II Glutamate–cysteine ligase

v t e Enzymes
Activity	Active site Binding site Catalytic triad Oxyanion hole Enzyme promiscuity Diffusion-limited enzyme Cofactor Enzyme catalysis
Regulation	Allosteric regulation Cooperativity Enzyme inhibitor Enzyme activator
Classification	EC number Enzyme superfamily Enzyme family List of enzymes
Kinetics	Enzyme kinetics Eadie–Hofstee diagram Hanes–Woolf plot Lineweaver–Burk plot Michaelis–Menten kinetics
Types	EC1 Oxidoreductases (list) EC2 Transferases (list) EC3 Hydrolases (list) EC4 Lyases (list) EC5 Isomerases (list) EC6 Ligases (list) EC7 Translocases (list)

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