δ-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine β-Synthase Expressed in Escherichia coli

We recently expressed human cystathionine β-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, CES, only about 20% saturated with heme, was purified from transformed bacteria. In the present study, we supplemented the bacteria with 0.3 mM δ-aminolevulinate (δALA), 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 δALA-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 δALA synthase. The δALA 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.