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PDBsum entry 2c8m
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References listed in PDB file
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Key reference
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Title
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Structure of a putative lipoate protein ligase from thermoplasma acidophilum and the mechanism of target selection for post-Translational modification.
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Authors
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E.Mcmanus,
B.F.Luisi,
R.N.Perham.
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Ref.
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J Mol Biol, 2006,
356,
625-637.
[DOI no: ]
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PubMed id
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Abstract
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Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo
acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate
protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in
Escherichia coli. The recombinant protein, a monomer of molecular mass 29kDa,
was catalytically inactive. Crystal structures in the absence and presence of
bound lipoic acid were solved at 2.1A resolution. The protein was found to fall
into the alpha/beta class and to be structurally homologous to the catalytic
domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA.
Lipoic acid in LplA was bound in the same position as biotin in BirA. The
structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA
together highlighted some key features of the post-translational modification. A
loop comprising residues 71-79 in the T.acidophilum ligase is proposed as
interacting with the dithiolane ring of lipoic acid and discriminating against
the entry of biotin. A second loop comprising residues 179-193 was disordered in
the T.acidophilum structure; tryptic cleavage of the corresponding loop in the
E.coli LplA under non-denaturing conditions rendered the enzyme catalytically
inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a
C-terminal domain found in its counterparts in E.coli (Gram-negative) or
Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears
to have structural homology to the additional domain in the E.coli and
S.pneumoniae enzymes was detected alongside the structural gene encoding the
putative LplA in the T.acidophilum genome. It is likely that this protein is
required to confer activity on the LplA as currently purified, one protein
perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and
the other transferring the lipoyl group from it to the specific lysine residue
in the target protein.
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Figure 2.
Figure 2. Structure of the LplA of T. acidophilium with
lipoic acid bound at the active site. The red colouring denotes
the boundaries of the disordered region that comprises residues
179-193. The lipoic acid is depicted in sticks and designated
with an arrowhead. The helices of interest are numbered H1 and
H2; the b-strands of interest are numbered B4, B7 and B8.
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Figure 3.
Figure 3. Lipoic acid binding in the LplA of T.
acidophilum. (a) 2F[o] -F[c] map of electron density from a
crystal of the native LplA soaked with R,S-lipoic acid. Lipoic
acid can be seen forming interactions with Arg72 which itself
forms a hydrogen bond with the carbonyl oxygen of Gly77. This
interaction is shown by the broken red lines. The bond distances
to the carbonyl oxygen are 3.06 and 2.86 Å. (b) Amino acid
residues in the lipoic acid binding site that are highly
conserved in LplAs. The residue numbers are shown in black using
T. acidophilum LplA numbering. (c) Comparison of the proposed
lipoic acid-binding sites in the T. acidophilum and E. coli
lipoate protein ligases. The T. acidophilum protein and lipoic
acid are coloured green whereas the E. coli protein and lipoic
acid are coloured blue.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
356,
625-637)
copyright 2006.
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