BirA binds as a dimer to its 40 bp DNA site, the biotin operator [2]. An additional, low-affinity BirA DNA binding site has been identified [12]. Initial models suggested that monomeric BirA-bio-5'-AMP shows cooperative binding to its DNA site [10], whereas more recent studies indicate that dimerization occurs before DNA binding [13]. BirA-BCCP binding may preclude dimerization and therefore DNA binding and repressor activity [14]. Biotinyl-5'-adenylate (bio-5'-AMP, the physiological effector) [15] and biotin [16, 17] stimulate dimerization and DNA binding. Crystal structures at 2.3 angstrom resolution [7] and of a BirA-biotin complex [16] are presented. Crystallization has been described [18]. The implications of the structure with respect to the binding of biotin and ATP are discussed [7]. ApoBirA, the BirA-bio-5'-AMP complex, and the BirA-biotin complex have distinct structural characteristics [9, 19]. DNA binding affects the structure of the C terminus as well as the structure of the N terminus [19]. Disordered loop structures on the protein surface appear to be involved in binding to biotin, bio-5'-AMP, and/or DNA [19] and in protein dimerization [16, 20]. A model of binding and reaction progression is presented [21]. The enzymatic reaction of bio-5'-AMP formation has been kinetically characterized [22], and the kinetics of biotinylation of the biotin carboxyl carrier protein monomer have been determined [23]. The thermodynamics of association between BirA and biotin and between BirA and bio-5'-AMP have been characterized [8, 21], as have the thermodynamics of dimerization and DNA binding by BirA, the BirA-biotin complex, and the BirA-bio-5'-AMP complex [24]. Substrate characteristics have been examined [25, 26, 27, 28, 29, 30, 31]. Superrepressor biotin binding and homodimerization properties were measured and showed that all variants exhibit biotin binding affinities similar to that measured for the BirA wild type. Altered dimerization results in changes in an electrostatic network that contribute to allosteric activation of BirA for dimerization [32] which occurs via coupled distant disorder-to-order transitions [33]. Combined structural and computational studies showed that BirA is part of an extensive residue network, and substitution of several network residues yields large perturbations to allostery [33] Deletion of the N-terminal DNA binding domain eliminates DNA binding activity and reduces binding to biotin and bio-5'-AMP, but does not affect the production of bio-5'-AMP or the BCCP biotinylation activity [34]. The DNA binding region is distinct from the regions in which mutations lead to heat sensitivity [35]. G115S, R118G, and R119W mutations cause defects in binding to biotin and bio-5'-AMP [36]. Mutations in the region that binds bio-5'-AMP also cause defects in dimerization and DNA binding [20]. An R317E mutation affects binding to ATP, and a K277E mutation affects substrate recognition [37]. BirA overproduction and purification is described [6]. BirA fusion proteins with affinity tags have been purified [38, 39]. Structural similarity between lipoylating and biotinylating enzymes, and implications with respect to the catalytic site, are discussed [40]. BirA has been used as a reagent for biotinylation of exogenous proteins [26, 41, 42, 43, 44, 45, 46, 47]. birA mutants contain high levels of biotin biosynthetic enzymes, overproduce and secrete biotin [48, 49, 50], and exhibit resistance to α-dehydrobiotin [50]. Some birA mutations cause heat sensitivity [51]. Mutants also show defects in transcriptional repression at the biotin operon promoter [52] and an increased requirement for biotin [53]. BirA: "biotin retention" [48] BioR: "biotin regulator" [54] Reviews: [1, 55, 56, 57, 58, 59]