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, CES, 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. (C) 1995 Academic Press, Inc.