Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

Abstract

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed beta- and gamma-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the alpha- and beta-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg(2+), and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPDelta67 fragment and BSA, and is subject to self-biotinylation.

Early evolution of the biotin-dependent carboxylase family

“…As previously mentioned, two main types of BPL can be found in prokaryotes: the so-called BPL that solely bears a BPL function, and BirA, that has a DNA-binding domain in its N-terminus responsible for a transcriptional regulatory role. Although quaternary structure differences among different taxa have been major concerns in previous literature [23,24,50,51], we will only consider here the major distinction concerning the bifunctionality of BirA with respect to the monofunctionality of BPL [22]. Accordingly, we classified prokaryotic sequences into the BPL or BirA groups on the basis of the absence or presence of the DNA-binding N-terminal domain, respectively.…”

Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus

“…In other BPLs these features, known as the biotin binding loop (BBL) and ATP binding loop (ABL), are not visible in the unliganded form of the enzyme but are observed when ligand is bound. The disordered-to-ordered transition that accompanies ligand binding has been previously reported (33)(34)(35)(36). In the complex of SaBPL with 2, the BBL folds against the central ␤-sheet in the central domain to cover the active site and maintain the reaction intermediate in situ (Fig.…”

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

“…The 7 N-terminus residues and two loop regions between residues 65–76 (L4) and 162–169 (L8) are not there in both subunits of high (1.8 Å, 2cgh) and low-resolution structures (2.8 Å, hMtb-BirA). The disordered loops are undetectable in other BirA structures (1bia, 1wq7 and 3fjp) as well and are associated with the conformational changes upon biotinyl-5′-AMP binding [12]–[14].…”

“…Several crystal structures of both monofunctional and bifunctional BirAs [8], [9] from many genera have been determined either as apoenzyme or as complex with its ligands (Table 1) [10]–[14]. All the apo BirA crystal structures have revealed the presence of disordered flexible loops, which undergo a conformational transition upon biotin and biotinyl-5′-AMP binding.…”