Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.
The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.
Schurr
M J
MJ
Department of Biological Sciences, University of North Texas, Denton 76203.
Vickrey
J F
JF
Kumar
A P
AP
Campbell
A L
AL
Cunin
R
R
Benjamin
R C
RC
Shanley
M S
MS
O'Donovan
G A
GA
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004080:Dihydroorotase / Q000235:genetics / Q000502:physiology*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment
D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase