THE AMINO-ACID-SEQUENCE OF RAT-KIDNEY 5-OXO-L-PROLINASE DETERMINED BYCDNA CLONING

5-Oxoprolinase (EC 3.5.2) catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exe rgonic hydrolysis of ATP to ADP and inorganic phosphate, Highly purifi ed preparations of the enzyme have been obtained from rat kidney and P seudomonas putida, The rat kidney enzyme is composed of two strongly i nteracting, apparently identical subunits (M(r) = 142,000), whereas th at from P. putida is composed of two functionally different protein co mponents that can readily be dissociated, Here we report the cloning o f rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a lambda gt11 cD NA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme, The whole cDN A clone was completed by amplifying its 5' end from a premade library of rat kidney Marathon-Ready(TM) cDNAs using polymerase chain reaction methodology, The composite cDNA (4,016 bases) revealed an uninterrupt ed open reading frame encoding 1,288 amino acid residues (M(r) = 137,7 59), The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived f rom the enzyme, Expression of the full-length clone in Escherichia col i yielded a product of the same size as the rat kidney enzyme and whic h reacted with antibodies directed against the rat kidney enzyme, The predicted amino acid sequence is almost 50% identical throughout its e ntire length to that of a hypothetical yeast protein YKL215C, It is al so 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase Hyu B, The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common pr operty of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.