Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue.
The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.
Russell
G C
GC
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Guest
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D006639:Histidine; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping
D003062:Codon; D009841:Oligonucleotides; D011768:Pyruvate Dehydrogenase Complex; D006639:Histidine