title: Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP.
abstract: Lipoic acid is an essential cofactor of the alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. It is covalently attached to a specific lysine residue of the subunit of the complexes. The bovine lipoyltransferase (bLT) catalyzes the lipoic acid attachment reaction using lipoyl-AMP as a substrate, forming a lipoylated protein and AMP. To gain insights into the reaction mechanism at the atomic level, we have determined the crystal structure of bLT at 2.10 A resolution. Unexpectedly, the purified recombinant bLT contains endogenous lipoyl-AMP. The structure of bLT consists of N-terminal and C-terminal domains, and lipoyl-AMP is bound to the active site in the N-terminal domain, adopting a U-shaped conformation. The lipoyl moiety is buried in the hydrophobic pocket, forming van der Waals interactions, and the AMP moiety forms numerous hydrogen bonds with bLT in another tunnel-like cavity. These interactions work together to expose the C10 atom of lipoyl-AMP to the surface of the bLT molecule. The carbonyl oxygen atom of lipoyl-AMP interacts with the invariant Lys135. The interaction might stimulate the positive charge of the C10 atom of lipoyl-AMP, and consequently facilitate the nucleophilic attack by the lysine residue of the lipoate-acceptor protein, accompanying the bond cleavage between the carbonyl group and the phosphate group. We discuss the structural differences between bLT and the lipoate-protein ligase A from Escherichia coli and Thermoplasma acidophilum. We further demonstrate that bLT in mitochondria also contains endogenous lipoylmononucleotide, being ready for the lipoylation of apoproteins.
authors.affiliation: Institute for Enzyme Research, the University of Tokushima, Tokushima 770-8503, Japan. fujiwara@ier.tokushima-u.ac.jp
authors.forename: Kazuko
authors.initials: K
authors.lastname: Fujiwara
authors.forename: Harumi
authors.initials: H
authors.lastname: Hosaka
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsuda
authors.forename: Kazuko
authors.initials: K
authors.lastname: Okamura-Ikeda
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Motokawa
authors.forename: Mamoru
authors.initials: M
authors.lastname: Suzuki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakagawa
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013822:Thermoplasma / Q000201:enzymology
chemical_list: D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000217:Acyltransferases