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.
Kery
V
V
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Elleder
D
D
Kraus
J P
JP
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