Hepatic carbamoyl phosphate synthetase (CPS) I and urea contents in the hylid tree frog, Litoria caerulea: transition from CPS III to CPS I
The complete cDNA sequence of CPS I obtained from the liver of the hylid tree frog, Litoria caerulea, consisted of 4,485 bp which coded for 1,495 amino acids with an estimated molecular mass of 163.7 kDa. The deduced CPS I consisted of a mitochondrial targeting sequence of 33 amino acid residues, a glutaminase amidotransferase component spanning from tyrosine 95 to leucine 425, and a methylglyoxal synthetase-like component spanning from valine 441 to lysine 1566. It also comprised two cysteine residues (cysteine 1360 and cysteine 1370) that are characteristic of N-acetyl-l-glutamate dependency. Similar to the CPS I of Ranacatesbeiana and Cps III of lungfishes and teleosts, it contained the Cys–His–Glu catalytic triad (cysteine 304, histidine 388 and glutamate 390). All Cps III contain methionine 305 and glutamine 308, which are essential for the Cys–His–Glu triad to react with glutamine, but the CPS I of R.catesbeiana contains lysine 305 and glutamate 308, and therefore cannot effectively utilize glutamine as a substrate. However, the CPS I of L. caerulea, unlike that of R.catesbeiana, contained besides glutamate 308, methionine 305 instead of lysine 305, and thus represented a transitional form between Cps III and CPS I. Indeed, CPS I of L. caerulea could utilize glutamine or NH4+ as a substrate in vitro, but the activity obtained with glutamine + NH4+ reflected that obtained with NH4+ alone. Furthermore, only <5 % of the glutamine synthetase activity was present in the hepatic mitochondria, indicating that CPS I of L. caerulea did not have an effective supply of glutamine in vivo. Hence, our results confirmed that the evolution of CPS I from Cps III occurred in amphibians. Since L. caerulea contained high levels of urea in its muscle and liver, which increased significantly in response to desiccation, its CPS I had the dual functions of detoxifying ammonia to urea and producing urea to reduce evaporative water loss.