Biotin—(acetyl-CoA-carboxylase) ligase


Biotin— 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 by performing two distinct functions: it acts as a biotin protein ligase, catalyzing the covalent attachment of biotin to its target proteins, and as a transcriptional repressor, controlling the expression of the biotin biosynthesis operon.
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 -, 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.

Nomenclature and Classification

The systematic name of this enzyme class is biotin:apo- ligase . Other names in common use include:
It belongs to the family of ligases, specifically those forming carbon-nitrogen bonds as acid-D-amino-acid ligases.

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, a subunit of the enzyme Acetyl-CoA Carboxylase.
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 Holo-BCCP + AMP + PPi
Without a functional BirA, BCCP remains in its apo- 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, 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.
  • 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 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:
  1. N-terminal Domain: Contains a classic helix-turn-helix DNA-binding motif. In the apo-enzyme, this domain is highly flexible and disordered.
  2. 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.
  3. 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:
BirA uses ATP to activate the carboxyl group of biotin, forming a high-energy mixed anhydride intermediate, biotinyl-5'-adenylate, and releasing pyrophosphate.
Biotin + ATP ⇌ Biotinyl-5'-AMP + PPi
The activated biotinyl group is transferred from AMP to the ε-amino group of the specific target lysine residue on the acceptor protein. 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. By genetically fusing the AviTag to a protein of interest, researchers can use BirA to specifically biotinylate that protein at a single, known site.