Expression of mammalian 5-aminolevulinate synthase in Escherichia coli. Overproduction, purification, and characterization.
5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D. S., and Curtis, P.J. (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein. Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate. A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture. Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml. Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources. These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor. Removal of the pyridoxal 5'-phosphate led to complete loss of activity. However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate. The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme. This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E. coli and its purification from an overproducing strain. The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.
Ferreira
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GC
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612-4799.
Dailey
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HA
D000624:5-Aminolevulinate Synthetase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000624:5-Aminolevulinate Synthetase