Evolution of vertebrate glucokinase regulatory protein from a bacterial N-acetylmuramate 6-phosphate etherase

Abstract

Mammalian GKRP [GK (glucokinase) regulatory protein], a fructose 6-phosphate and fructose 1-phosphate sensitive inhibitor of GK, appears to have resulted from the duplication of a gene similar to bacterial N-acetylmuramate 6-phosphate etherase MurQ. In the present study, we show that several genomes of primitive eukaryotes encode a GKRP-like protein with two MurQ repeats. Recombinant Haemophilus influenzae MurQ and the GKRP homologue of the amoeboflagellate Naegleria gruberi both behaved as excellent N-acetylmuramate 6-phosphate etherases, with Kcat values (83 and 20 s−1) at least as high as that reported for Escherichia coli MurQ. In contrast, rat and Xenopus GKRP displayed much lower etherase activities (Kcat=0.08 and 0.05 s−1 respectively). The etherase activity of rat GKRP was inhibited by ligands (fructose 6-phosphate, fructose 1-phosphate and sorbitol 6-phosphate) known to regulate its interaction with GK and by mutations affecting the binding of these phosphate esters. This indicated that these phosphate esters all bind to a single regulatory site, which evolved from the original catalytic site. Sorbitol 6-phosphate and other phosphate esters also inhibited the etherase activity of Xenopus GKRP, but did not affect its ability to inhibit GK. Thus, unlike what was previously thought, Xenopus GKRP has a binding site for phosphate esters, but this site is uncoupled from the GK-binding site. Taken together, these data indicate that duplication of the murQ gene led to a eukaryotic-type etherase, which subsequently evolved to GKRP by acquiring a new binding specificity while losing most of its etherase activity.