Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase.
Enterococcus faecalis lipoamidase was discovered almost 50 years ago (Reed, L. J., Koike, M., Levitch, M. E., and Leach, F. R. (1958) J. Biol. Chem. 232, 143-158) as an enzyme activity that cleaved lipoic acid from small lipoylated molecules and from pyruvate dehydrogenase thereby inactivating the enzyme. Although the partially purified enzyme was a key reagent in proving the crucial role of protein-bound lipoic acid in the reaction mechanism of the 2-oxoacid dehydrogenases, the identity of the lipoamidase protein and the encoding gene remained unknown. We report isolation of the lipoamidase gene by screening an expression library made in an unusual cosmid vector in which the copy number of the vector is readily varied from 1-2 to 40-80 in an appropriate Escherichia coli host. Although designed for manipulation of large genome segments, the vector was also ideally suited to isolation of the gene encoding the extremely toxic lipoamidase. The gene encoding lipoamidase was isolated by screening for expression in E. coli and proved to encode an unexpectedly large protein (80 kDa) that contained the sequence signature of the Ser-Ser-Lys triad amidohydrolase family. The hexa-histidine-tagged protein was expressed in E. coli and purified to near-homogeneity. The purified enzyme was found to cleave both small molecule lipoylated and biotinylated substrates as well as lipoic acid from two 2-oxoacid dehydrogenases and an isolated lipoylated lipoyl domain derived from the pyruvate dehydrogenase E2 subunit. Lipoamidase-mediated inactivation of the 2-oxoacid dehydrogenases was observed both in vivo and in vitro. Mutagenesis studies showed that the residues of the Ser-Ser-Lys triad were required for activity on both small molecule and protein substrates and confirmed that lipoamidase is a member of the Ser-Ser-Lys triad amidohydrolase family.
Jiang
Yanfang
Y
Department of Microbiology, the University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Cronan
John E
JE
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D008969:Molecular Sequence Data; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D000581:Amidohydrolases