Expression of cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase in Escherichia coli. Differences in kinetic mechanisms.
The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in Escherichia coli using a lambda PL promoter-driven prokaryotic expression vector. The expressed proteins in total cell extracts were identified by Western blot analysis using anti-pig heart E3 antibody and also by measurement of E3 activity. Most of the expressed human E3 polypeptides (five bands) were found in the insoluble pellet while primarily full-length mature E3 was found in the soluble fraction. About 2% of the total soluble protein was mature human E3 when expressed in wild type E. coli AR120. Since wild type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli, JRG1342. The expressed recombinant human E3s in JRG1342 were purified to near homogeneity. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 had an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about half of the mature E3 activity. The double-reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. These kinetic results showed that the partial 16-amino acid extension in the leader sequence changed the kinetic mechanism of human E3 so that it resembled that of glutathione reductase.
Kim
H
H
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Liu
T C
TC
Patel
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics*; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics*
D011498:Protein Precursors; D004247:DNA; D008058:Dihydrolipoamide Dehydrogenase