Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis

J. D. Moore, H. K. Lamb, T. Garbe, S. Servos, G. Dougan, I. G. Charles, A. R. Hawkins

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29 Citations (Scopus)

Abstract

The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable stable, is active as a homododecamer and has the lowest reported K(m) value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA quiB double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE mutant strain of A. nidulans to utilize quinate as sole carbon source.

Original languageEnglish
Pages (from-to)173-181
Number of pages9
JournalBiochemical Journal
Volume287
Issue number1
DOIs
Publication statusPublished - 1 Jan 1992
Externally publishedYes

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