OXP1/YKL215c encodes an ATP-dependent 5-oxoprolinase in Saccharomyces cerevisiae: functional characterization, domain structure and identification of actin-like ATP-binding motifs in eukaryotic 5-oxoprolinases

Abstract

OXP1/YKL215c, an uncharacterized ORF of Saccharomyces cerevisiae, encodes a functional ATP-dependent 5-oxoprolinase of 1286 amino acids. The yeast 5-oxoprolinase activity was demonstrated in vivo by utilization of 5-oxoproline as a source of glutamate and OTC, a 5-oxoproline sulfur analogue, as a source of sulfur in cells overexpressing OXP1. In vitro characterization by expression and purification of the recombinant protein in S. cerevisiae revealed that the enzyme exists and functions as a dimer, and has a K_m of 159 μM and a V_max of 3.5 nmol h^-1μg^-1 protein. The enzyme was found to be functionally separable in two distinct domains. An 'actin-like ATPase motif' could be identified in 5-oxprolinases, and mutation of key residues within this motif led to complete loss in ATPase and 5-oxoprolinase activity of the enzyme. The results are discussed in the light of the previously postulated truncated γ-glutamyl cycle of yeasts.