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J. Biol. Chem., Vol. 279, Issue 39, 40345-40350, September 24, 2004

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Heterooligomeric Phosphoribosyl Diphosphate Synthase of Saccharomyces cerevisiae

COMBINATORIAL EXPRESSION OF THE FIVE PRS GENES IN ESCHERICHIA COLI^*

Bjarne Hove-Jensen

From the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1307 Copenhagen, Denmark

The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1–5), which specify PRPP synthase subunits 1–5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (prs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on P_i, to be temperature-sensitive, and inhibited by ADP.

Received for publication, May 17, 2004 , and in revised form, July 20, 2004.

^* This work was supported by the Danish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) X75075 (PRS2) and AJ245726 (PRS4)

To whom correspondence should be addressed: Dept. of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K, Denmark. Tel.: 45-3532-2027; Fax: 45-3532-2040; E-mail: hove{at}mermaid.molbio.ku.dk.

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