Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli: DETERMINATION OF THE LIPOIC ACID-BINDING SITE

Kazuko Fujiwara,Sachiko Toma,Kazuko Okamura-Ikeda,Yutaro Motokawa,Atsushi Nakagawa,Hisaaki Taniguchi

doi:10.1074/jbc.m505010200

Kazuko Fujiwara, Sachiko Toma + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m505010200

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Abstract

Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.

Highlights

Lipoic acid is a prosthetic group of acyltransferase (E2) subunit of the pyruvate, ␣-ketoglutarate, and branched-chain ␣-ketoacid dehydrogenase complexes and of H-protein of the glycine cleavage system [1,2,3,4]
Strong homology in the N-terminal half region suggests that the region may be responsible for the second transfer reaction, whereas some residue(s) in the C-terminal region of the E. coli enzyme may be essential for the lipoate-activating reaction, which are lacking in the mammalian sequences [10]
Structure Determination—Crystallization of E. coli Lipoate-protein ligase A (LplA) was mentioned about 10 years ago [6]

Summary

EXPERIMENTAL PROCEDURES

Construction of the Expression Vector Containing lplA Gene—DNA manipulations were accomplished by the standard techniques [12]. The PCR product was inserted into the pET-3a expression vector [13]. The construct pET-LPLA was verified by DNA sequencing and transformed into E. coli expression strain BL21(DE3)pLysS or B834(DE3)pLysS to obtain native LplA or selenomethionine-substituted LplA (Se-LplA), respectively. The cell extract was obtained by centrifugation at 105,000 ϫ g for 1 h and applied onto a hydroxylapatite column (3 ϫ 6 cm) equilibrated with 3 mM potassium phosphate buffer, pH 7. 4, containing 0.5 mM DTT and 10 ␮M APMSF and further purified by a HiPrep 16/10 DEAE column (Amersham Biosciences) equilibrated with 20 mM Tris-HCl, pH 7. After the recombination reaction with pET-3a digested with NdeI and BamHI and the pair of the PCR products, the resultant plasmid was transformed into E. coli BL21(DE3)pLysS. The mutant LplAs were expressed and purified up to the DEAE-Sepharose

Ramachandran plot

RESULTS AND DISCUSSION

Varied substrate