ASPARTATE TRANSCARBAMOYLASE GENES OF PSEUDOMONAS-PUTIDA - REQUIREMENTFOR AN INACTIVE DIHYDROOROTASE FOR ASSEMBLY INTO THE DODECAMERIC HOLOENZYME

The nucleotide sequences of the genes encoding the enzyme aspartate tr anscarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately fr om overlapping genes. The P. putida ATCase does not possess dissociabl e regulatory and catalytic functions but instead apparently contains t he regulatory nucleotide binding site within a unique N-terminal exten sion of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp lo ng and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the e nzyme. The second gene is 1,275 bp long and encodes a 424-residue poly peptide which bears significant homology to dihydroorotase (DHOase) fr om other organisms. Despite the homology of the overlapping gene to kn own DHOases, this 44.2-kDa polypeptide is not considered to be the fun ctional product of the pyrC gene in P. putida, as DHOase activity is d istinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide la cks specific histidyl residues thought to be critical for DHOase enzym atic function. The pyrC-like gene (henceforth designated pyrC') does n ot complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the ve stigial DHOase is to maintain ATCase activity by conserving the dodeca meric assembly of the native enzyme. This unique assembly of six activ e pyrB polypeptides coupled with six inactive pyrC' polypeptides has n ot been seen previously for ATCase but is reminiscent of the fused tri functional CAD enzyme of eukaryotes.