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.
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 microM and a V(max) of 3.5 nmol h(-1) microg(-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 gamma-glutamyl cycle of yeasts.
Kumar
Akhilesh
A
Institute of Microbial Technology (CSIR), Chandigarh, India.
Bachhawat
Anand Kumar
AK
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D015870:Gene Expression; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011752:Pyroglutamate Hydrolase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D013455:Sulfur; D000255:Adenosine Triphosphate; D011752:Pyroglutamate Hydrolase; D000251:Adenosine Triphosphatases; D011761:Pyrrolidonecarboxylic Acid