Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase. 1552 86

Cancer immunotherapy relies on the identification and characterization of tumour antigens that can be recognized by effector T cells. Here, we used a proteomics-based approach to identify tumour antigens recognized by serum antibodies from patients with breast cancer. Specific reactivity against a set of spots was identified and their identity was revealed by MALDI-TOF peptide mass fingerprinting. They include disintegrin and metalloprotease 10, aldolase A, beta-ATPase F1, heat shock protein 27, deaminase, pyruvate dehydrogenase protein X component, and Vimentin. Western blot analysis using recombinant proteins expressed in E. coli confirmed the specific reactivity with patient sera. Several tumour antigens were expressed on the surface of the T7 phage and shown to trigger specific immune responses in BALB/c mice following oral immunisation. Furthermore, these immune responses inhibited tumour growth and metastasis of the 4T1 mammary adenocarcinoma cell line. Collectively, the present data indicate that proteomics-based strategy can identify tumour antigens whose surface display on phages or bacteria can provide an effective strategy for mucosal cancer vaccines. In addition, arrayed phage-displayed tumour antigens could be useful as a serum-based screening test for the detection of several tumour antigens.
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PMID:Mucosal vaccination with phage-displayed tumour antigens identified through proteomics-based strategy inhibits the growth and metastasis of 4T1 breast adenocarcinoma. 1809 64