Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

Abstract

Background: The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.

Results: Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine.

Conclusion: Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation was gradual, from complete inhibition of bacterial NAGS, to partial inhibition of fish NAGS, to activation of frog and mammalian NAGS. This change also coincided with the conquest of land by amphibians and mammals.

Figure 1

Urea cycle in tetrapods, fish and invertebrates. The first three enzymes of the urea cycle are localized in the mitochondria; the remaining three enzymes are cytoplasmic. Mammals and amphibians (tetrapods) have CPSI, which catalyzes the formation of CP from ammonia, bicarbonate and ATP. NAG is an essential allosteric activator of CPSI. CPSIII catalyzes the formation of CP from glutamine, bicarbonate and ATP in fish and invertebrates. The enzymatic activity of CPSIII increases in the presence of NAG. Abbreviations: NAGS – N-acetylglutamate synthase; NAG – N-acetylglutamate; CPSI – carbamylphosphate synthetase I; CPSIII – carbamylphosphate synthetase III; OTC – ornithine transcarbamylase; ASS – argininosuccinate synthase; ASL – argininosuccinate lyase.

Figure 2

Conservation of amino acid sequences of NAGS from 25 organisms. The sizes of letters indicate the degree of conservation. Residues that are important for arginine binding are highlighted in yellow. Invariant residues are shown in blue. Asterisks indicate invariant residues that are mutated in arginine-insensitive NAGS from E. coli. Arrows indicate conserved amino acids that are absent or replaced by other amino acids in E. coli NAGK, which is not inhibited by arginine. Amino acids that were mutated in this study are shown in red; mutations in the mouse NAGS are shown in green; mutations in the X. campestris NAGS-K are in purple. LOGO-alignment was generated using NAGS sequences from five mammals (human, mouse, rat, dog and cow), two amphibians (X. laevis and X. tropicalis), zebrafish, pufferfish, freshwater pufferfish, arabidopsis, soy, tomato, rice, corn and 11 bacteria (E. coli, R. eutropha, N. gonorrhoeae, P aeruginosa, P. syringiae, X. campestris, X. axonopodis, X. fastidiosa, P. bermudensis, O. alexandrii and M. maris), which were aligned using ClustalW.

Figure 3

Purification of wild-type and arginine-insensitive NAGS. Wild-type and mutant mouse NAGS (A) and X. campestris NAGS-K (B) were overexpressed in E. coli and purified using nickel-affinity chromatography.

Figure 4

Distribution of the ability to synthesize urea, CPSI and CPSIII in Deuterostomes. Animals whose genomes were surveyed in this study are indicated with asterisks. All six urea cycle genes were identified in the genomes of zebrafish, pufferfish, freshwater pufferfish and sea urchin, indicating potential ability of these animals to synthesize urea. Full sets of urea cycle genes were not found in the genomes of sea squirts C. intestinalis and C. savygnii. Numbers in parentheses are numbers in the reference list. The cladogram indicates taxonomic relationships among phyla; the length of each clade does not indicate evolutionary distance between phyla.

Figure 5

Purification of recombinant NAGS from vertebrates, plant and bacteria. Each protein had N-terminal polyhistidine affinity tag, was overexpressed in E. coli and purified using nickel-affinity chromatography.