Abstract
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 exergonic hydrolysis of ATP to ADP and inorganic phosphate. Highly purified preparations of the enzyme have been obtained from rat kidney and Pseudomonas putida. The rat kidney enzyme is composed of two strongly interacting, apparently identical subunits (Mr = 142,000), whereas that from P. putida is composed of two functionally different protein components that can readily be dissociated. Here we report the cloning of rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a lambdagt11 cDNA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme. The whole cDNA clone was completed by amplifying its 5' end from a premade library of rat kidney Marathon-ReadyTM cDNAs using polymerase chain reaction methodology. The composite cDNA (4,016 bases) revealed an uninterrupted open reading frame encoding 1,288 amino acid residues (Mr = 137,759). The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived from the enzyme. Expression of the full-length clone in Escherichia coli yielded a product of the same size as the rat kidney enzyme and which reacted with antibodies directed against the rat kidney enzyme. The predicted amino acid sequence is almost 50% identical throughout its entire length to that of a hypothetical yeast protein YKL215C. It is also 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase HyuB. The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common property of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, NY 10021
The amino acid sequences corresponding to these four peptides were subsequently found in the cDNA clone (Fig. 2)
Though CL273 was the largest (3.2 kb) and the most 5Ј-extended, this clone was found to be an artifact at its 5Ј region on the basis of restriction mapping and sequencing; i.e. about two-thirds of the 5Ј terminal region of CL273 was from an unknown cDNA clone, as judged in part by the presence of a poly(A) tail in this region
Summary
§ To whom correspondence should be addressed: Dept. of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Homogeneous preparations of 5-OPase from rat kidney [6] and Pseudomonas putida [7] had been obtained in this laboratory and were used for physical characterization and for studies of catalytic mechanism. Knowledge of the amino acid sequence of the enzyme and the cloning of the encoding cDNA are essential for further studies on the structure, mechanism of action, and physiological function of the enzyme. This study was undertaken to elucidate the primary structure of rat kidney 5-OPase as preparation for the analysis of relationships between its structure and function. Peptides obtained from the purified rat kidney enzyme by enzymatic cleavage with Lys-C were used to design oligonucleotide probes that permitted cloning and sequencing of the cDNA encoding the complete protein. Protein representing the composite whole cDNA and truncated cDNA clones of the enzyme was expressed in Escherichia coli, as confirmed by immunodetection with the rabbit antiserum against isolated rat kidney 5-OPase
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