Delta-aminolevulinate increases heme saturation and yield of human cystathionine beta-synthase expressed in Escherichia coli.
We recently expressed human cystathionine beta-synthase (CBS) in Escherichia coli and purified it to homogeneity. We showed that CBS requires heme in addition to pyridoxal 5'-phosphate for its function. Previously, CBS, only about 20% saturated with heme, was purified from transformed bacteria. In the present study, we supplemented the bacteria with 0.3 mM delta-aminolevulinate (delta ALA), a precursor of heme. While growth of the bacteria did not change, a 50-fold elevation of the heme content per milligram of total protein was observed in the cell extracts of delta ALA-supplemented cells. The increase in heme biosynthesis depended on the overexpression of a heme acceptor--CBS. Our data suggest that bacterial heme synthesis is regulated beyond delta ALA synthase. The delta ALA treatment resulted in 8 times more total CBS activity with a 3.5-fold higher yield of the purified recombinant enzyme, more than 68% saturated with heme. Increased yield, higher specific activity, and improved heme saturation of CBS will facilitate large-scale preparation of the enzyme. This method should be applicable to the overexpression of other recombinant heme proteins in bacteria.
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
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Kery
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Elleder
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Kraus
D000622:Aminolevulinic Acid / Q000494:pharmacology*; D003541:Cystathionine beta-Synthase / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000187:drug effects; D013053:Spectrophotometry; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D006420:Hemeproteins; D011993:Recombinant Fusion Proteins; D006418:Heme; D000622:Aminolevulinic Acid; D001616:beta-Galactosidase; D003541:Cystathionine beta-Synthase