Abstract
BackgroundIn the 1950s, Reed and coworkers discovered an enzyme activity in Streptococcus faecalis (Enterococcus faecalis) extracts that inactivated the Escherichia. coli and E. faecalis pyruvate dehydrogenase complexes through cleavage of the lipoamide bond. The enzyme that caused this lipoamidase activity remained unidentified until Jiang and Cronan discovered the gene encoding lipoamidase (Lpa) through the screening of an expression library. Subsequent cloning and characterization of the recombinant enzyme revealed that lipoamidase is an 80 kDa protein composed of an amidase domain containing a classic Ser-Ser-Lys catalytic triad and a carboxy-terminal domain of unknown function. Here, we show that the amidase domain can be used as an in vivo probe which specifically inactivates lipoylated enzymes.Methodology/Principal FindingsWe evaluated whether Lpa could function as an inducible probe of α-ketoacid dehydrogenase inactivation using E. coli as a model system. Lpa expression resulted in cleavage of lipoic acid from the three lipoylated proteins expressed in E. coli, but did not result in cleavage of biotin from the sole biotinylated protein, the biotin carboxyl carrier protein. When expressed in lipoylation deficient E. coli, Lpa is not toxic, indicating that Lpa does not interfere with any other critical metabolic pathways. When truncated to the amidase domain, Lpa retained lipoamidase activity without acquiring biotinidase activity, indicating that the carboxy-terminal domain is not essential for substrate recognition or function. Substitution of any of the three catalytic triad amino acids with alanine produced inactive Lpa proteins.Conclusions/SignificanceThe enzyme lipoamidase is active against a broad range of lipoylated proteins in vivo, but does not affect the growth of lipoylation deficient E. coli. Lpa can be truncated to 60% of its original size with only a partial loss of activity, resulting in a smaller probe that can be used to study the effects of α-ketoacid dehydrogenase inactivation in vivo.
Highlights
The cofactor lipoic acid is necessary for the oxidative decarboxylation of 2-ketoacids, an activity essential to the tricarboxylic acid cycle, amino acid metabolism, heme biosynthesis, and other metabolic processes
Lpa is expressed in active form at both 37uC and 20uC, and its activity at a range of temperatures suggests that it could be used as a probe of in vivo lipoyl protein inactivation in organisms other than E. coli
To confirm that the decreased biotinylation of the biotincarboxyl-carrier protein (BCCP) was derived from protein over-expression and was not Lpa-specific, we evaluated BCCP biotin levels after the expression of two proteins: P. falciparum malonyl-CoA:ACP acyltransferase (MCAT) and P. falciparum ketoacyl-ACP synthase II (KASII)
Summary
The cofactor lipoic acid is necessary for the oxidative decarboxylation of 2-ketoacids, an activity essential to the tricarboxylic acid cycle, amino acid metabolism, heme biosynthesis, and other metabolic processes. Oxidative decarboxylation is carried out by large, multisubunit complexes, including pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (KDH), and branched chain a-ketoacid dehydrogenase (BCDH) [1,2]. These complexes are composed of three subunits, the E1 a-ketoacid dehydrogenase, the E2 transacetylase, and the E3 dihydrolipoyldehydrogenase [1,3]. Lipoic acid plays two critical roles in a-ketoacid dehydrogenase complexes It is reductively acylated by the E1 subunit and subsequently functions as a swinging arm to transfer the covalently bound acyl group to the active site of the E3 subunit [1]. We show that the amidase domain can be used as an in vivo probe which inactivates lipoylated enzymes
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