Identification and Solution Structures of a Single Domain Biotin/Lipoyl Attachment Protein from Bacillus subtilis

Abstract

Protein biotinylation and lipoylation are post-translational modifications, in which biotin or lipoic acid is covalently attached to specific proteins containing biotin/lipoyl attachment domains. All the currently reported natural proteins containing biotin/lipoyl attachment domains are multidomain proteins and can only be modified by either biotin or lipoic acid in vivo. We have identified a single domain protein with 73 amino acid residues from Bacillus subtilis strain 168, and it can be both biotinylated and lipoylated in Escherichia coli. The protein is therefore named as biotin/lipoyl attachment protein (BLAP). This is the first report that a natural single domain protein exists as both a biotin and lipoic acid receptor. The solution structure of apo-BLAP showed that it adopts a typical fold of biotin/lipoyl attachment domain. The structure of biotinylated BLAP revealed that the biotin moiety is covalently attached to the side chain of Lys 35 , and the bicyclic ring of biotin is folded back and immobilized on the protein surface. The biotin moiety immobilization is mainly due to an interaction between the biotin ureido ring and the indole ring of Trp 12 . NMR study also indicated that the lipoyl group of the lipoylated BLAP is also immobilized on the protein surface in a similar fashion as the biotin moiety in the biotinylated protein.The biotin/lipoyl attachment domain (IPR000089) is a signature structural motif, which has a conserved lysine residue that can bind biotin or lipoic acid. This domain can be found in enzymes that require biotin or lipoic acid as cofactor (1). Biotin plays a catalytic role in carboxyl transfer reactions. It is covalently attached to a lysine residue, via an ⑀-amino group, in enzymes such as pyruvate carboxylase, acetyl-CoA carboxylase, and propionyl-CoA carboxylase, etc. (2). This process, called biotinylation, is catalyzed by biotin-protein ligase (BPL) 2 (1, 3).Lipoic acid can serve as cofactor in a variety of proteins, such as E2 acyltransferases (E2p) in the pyruvate dehydrogenase complex and H-protein of the glycine cleavage system (4, 5). It is also covalently bound via an amide linkage to a lysine group. This process, named lipoylation, is catalyzed by lipoate-protein ligase (LPL) (1, 6). It is known that BPLs have broad substrate ranges. Mammalian biotinyl domain can be biotinylated by bacterial BPL and vice versa. The situation for LPLs is also similar (1).The structures of several biotin/lipoyl attachment domains have been determined. Two of them are biotinyl domains, the C-terminal domain of Escherichia coli biotin carboxyl carrier protein (BCCP) and the biotinyl domain of 1.3 S subunit of transcarboxylase from Propionibacterium shermanii (7-10). Several structures of lipoyl domains have also been reported (11-15). All biotinyl domains and lipoyl domains share a very similar overall fold, which is a flattened ␤-barrel formed by two ␤-sheets. The conserved lysine residue is located at the tip of a tight ␤-turn.Although biotinyl and lipoyl domains are quite similar in ...

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

“…Moreover, E. coli LipB was able to modify all of the B. subtilis LDs tested indicating that the acceptor proteins were properly folded. However, the enigmatic biotin lipoyl acceptor protein (BLAP), a protein previously reported to be biotinylated and lipoylated when expressed in E. coli (Cui et al ., 2006) (Fig. 2) was not modified by any enzyme tested.…”

“…The B. subtilis BLAP was reported to be at least partially modified with lipoate when expressed in E. coli (Cui et al ., 2006). In that work the concentration of lipoic acid added to the culture medium corresponded to > 60 000‐fold excess over that needed for half‐maximal growth of a lipA strain (Reed and Cronan, 1993).…”

Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

“…B. subtilis has two known biotinylated proteins, pyruvate carboxylase (PyC) and the biotin carboxyl carrier protein (AccB) subunit of acetyl-CoA carboxylase (http://genodb.pasteur.fr). A third B. subtilis protein, biotin/lipoyl attachment protein (BLAP) encoded by the yngHB gene was found to be biotinylated and lipoylated when expressed in E. coli [22] although subsequently this protein was found not to be lipoylated by the B. subtilis enzymes that modify the known cognate lipoic acid acceptor proteins [23].…”

Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase

“…However, lipoate domains are structurally similar to biotin acceptor domains,46;47 and LplA is structurally related to biotin ligase as well 48. The co-crystal structure of P. horikoshii biotin ligase with its biotin acceptor protein shows a hydrogen bond between the +4 Glu of the acceptor and Lys27 of the enzyme 42.…”