Abstract
We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).
Highlights
Some purine nucleotides and nucleosides are commercially produced by fermentation [7, 23, 32]
E. coli is known to have inosine kinase activity [1, 15] which converts inosine to 5Ј-inosine monophosphate (5Ј-IMP), the precursor molecule of other nucleotides needed for cell growth
S⌽609 lacks the major purine nucleoside phosphorylase encoded by deoD [16], the inosine-hydrolyzing activity of the strain was still strong
Summary
Some purine nucleotides and nucleosides are commercially produced by fermentation [7, 23, 32]. Three enzymes are known to form 5Ј-IMP: hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), nucleoside phosphotransferase (EC 2.7.1.77), and inosine kinase (EC 2.7.1.73). Of these enzymes, inosine kinase is the least studied [2, 8]. It is difficult to correctly detect inosine kinase activity in crude extracts because of the other pathways that produce 5ЈIMP from inosine. Harlow et al [11] reported cloning of the gsk gene of E. coli by a strategy that was different from ours They showed that the sequence of their clone was almost identical to the sequence of our clone, and a crude extract of a strain in which gsk was overexpressed had both guanosine and inosine kinase activities. Our cloning strategy for the guanosine-inosine kinase gene and the unique character of the purified guanosineinosine kinase are described
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