EFFECT OF BIOSYNTHETIC MANIPULATION OF HOME ON INSOLUBILITY OF VITREOSCILLA HEMOGLOBIN IN ESCHERICHIA-COLI

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both so luble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli: Examination by atomic abso rption spectroscopy and electron paramagnetic resonance spectroscopy r evealed that the insoluble form uniformly lacks the heme prosthetic gr oup (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreosc illa cells. This observation suggested that a relationship may exist b etween the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemicall y and genetically manipulate the formation and conversion of 5-aminole vulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the interm ediate ALA and the competitive inhibitor levulinic acid which freely c ross the cell barrier. Genetic manipulations involved amplifying the g ene dosage for the enzymes ALA synthase and ALA dehydratase. Results f rom both levulinic aci;l and ALA supplementations indicate that the le vel of soluble holoVHb correlates with the heme level but that the lev el of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with he level of total VHb accumulated. The e ffect of amplifying ALA synthase and ALA dehydratase gene dosage is co mplex and may involve secondary factors. Results indicate that the rat e-limiting stet, of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli ( S. Hino and A. Ishida, Enzyme 16:42-49, 1973).