Hart, Roger A. and Kallio, Pauli T. and Bailey, James E. (1994) Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli. Applied and Environmental Microbiology, 60 (7). pp. 2431-2437. ISSN 0099-2240 http://resolver.caltech.edu/CaltechAUTHORS:HARaem94
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Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step 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).
|Additional Information:||Copyright © 1994 by the American Society for Microbiology. Received 15 December 1993/Accepted 10 May 1994. We thank Sharon Cosloy for providing the genes for ALA synthase and ALA dehydratase and Alexander Sassarman for helpful comments regarding heme biosynthesis in E. coli. This research was supported by the National Science Foundation (grant EET-8606179), by the Advanced Industrial Concepts Division of the U.S. Department of Energy, and by a grant for predoctoral training in biotechnology from the National Institute of General Medical Sciences (National Research Service Award 1 T32 GM 08346-01, Pharmacology Sciences Program).|
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|Deposited On:||23 Jun 2008|
|Last Modified:||26 Dec 2012 10:07|
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