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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 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A 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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SYNOPSIS. Sulfaquinoxaline and ethopabate are 2 chemically distinct types of antagonist of PABA with anticoccidial action. Reversal experiments with PABA and synergism studies with pyrimethamine indicated that both compounds interfered with the PABA-folk acid metabolic sequence. Six pure strains of Eimeriu brunetti responded differently to the 2 compounds. The strain most sensitive to ethopa-WO chemically distinct types of antimetabolite of p-T aminobenzoic acid (PABA) are recognized to have utility in the control of coccidiosis. Analogs of p-aminobenzenesulfonamide (sulfaquinoxaline, for example) represent one type. Nuclear-substituted derivatives of PABA were reported ( 6 , 7 ) to be potent anticoccidial compounds. Ethopabate (rnethyl-4-acetamino-2-ethoxybenzoate) is an example of this 2nd type of PABA antagonist.This paper reports biological comparisons between these 2 related coccidiostats. First, experiments are reported which characterize the 2 compounds as antagonists of PABA. After these initial studies, related to the mechanism of action, the objective was to extend the comparison between the 2 compounds to determine whether a number of isolates of coccidia were correspondingly sensitive to ethopabate and sulfaquinoxaline. A preliminary report of part of the data has been given(5).

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