Abstract
An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2' of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2' in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2'-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.
Highlights
An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate
Recombinant Expression of Dihydroneopterin Aldolases—We have recently reported the sequence of dihydroneopterintriphosphate epimerase from E. coli [15]
A data base search indicated that an open reading frame in the neighborhood of the bacA gene located at 69.0 kb on the E. coli chromosome specifies a similar protein (30% identical amino acid residues)
Summary
Materials—6-Hydroxymethylpterin, D-dihydroneopterin, L-dihydromonapterin, D-dihydromonapterin, L-neopterin, L-monapterin, D-dihydroneopterin monophosphate, D-neopterin, and dihydrobiopterin. Growth at 44 °C on plates that contain chloramphenicol selects clones with the plasmid integrated into the chromosome by homologous recombination This results in a tandem arrangement of the intact folX gene adjacent to the inactivated folX. The forward primer kan, complementary to the kanamycin gene, and the reverse primer Epi (Table II), complementary to the E. coli chromosome in the direct vicinity of the 3Ј-end of the folX insert, should result in the amplification of a 1.4-kb fragment. Purification of Recombinant Dihydroneopterin Aldolase—E. coli strain M15(pREP4) carrying the expression plasmid pEAL was grown to a density of 0.6 A600 nm units in shaking flasks with LB medium containing 20 g/ml kanamycin and 180 g/ml ampicillin at 37 °C.
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