View source for Biology:Carbamoyl phosphate synthetase III

{{short description|Protein isoform}}
'''Carbamoyl phosphate synthetase III''' (CPS III) is one of the three isoforms of the [[Biology:Carbamoyl phosphate synthetase|carbamoyl phosphate synthetase]], an enzyme that catalyzes the active production of [[Chemistry:Carbamoyl phosphate|carbamoyl phosphate]] in many organisms.

CPS III (EC 6.3.5.5.) is a [[Biology:Ligase|ligase]] (3.) that forms carbon-nitrogen bonds (6.3.) with [[Chemistry:Glutamine|glutamine]] as amido-N-donor (6.3.5.) (see [[Biology:BRENDA|BRENDA]]).

== Context ==
Many aquatic organisms, including most of the fish species, are ammoniotelic, which means they produce [[Chemistry:Ammonia|ammonia]] as [[Biology:Metabolic waste|metabolic waste]], that they generally excrete by diffusion through their [[Biology:Gill|gill]]s. Similar to terrestrial vertebrates, some fish species also significantly include [[Chemistry:Urea|urea]] as metabolic waste. This phenomenon concerns larvae stages since they do not have gills to excrete ammonia from, but also the adult stage in some species. Based on the proportion of metabolic waste represented by urea, these species are partial or fully ureotelic.

Ureotelic species produce urea via the ornithine-urea cycle (OUC) in which CPS plays an important role. [[Biology:Carbamoyl phosphate synthetase I|Carbamoyl phosphate synthetase I]] is mostly used by terrestrial vertebrates, and it appears that some aquatic species rely on CPS III to deal with urea production.<ref>{{cite journal|vauthors=Anderson PM |title=3 Urea Cycle in Fish: Molecular and Mitochondrial Studies|date= January 1995 |journal=Fish Physiology|volume=14|pages=57–83|veditors=Wood CM, Shuttleworth TJ |publisher=Academic Press|doi=10.1016/s1546-5098(08)60242-3 |isbn=9780123504388}}</ref> There are several potential advantages in excreting urea instead of ammonia for species living in specific environments. For example, it allows a better diffusion capacity than ammonia in alkaline waters,<ref name=":0">{{cite journal|vauthors=White LJ, Sutton G, Shechonge A, Day JJ, Dasmahapatra KK, Pownall ME |title=Adaptation of the carbamoyl-phosphate synthetase enzyme in an extremophile fish |journal=Royal Society Open Science |volume=7 |issue=10 |pages=201200 |date=October 2020 |pmid=33204476 |pmc=7657897 |doi=10.1098/rsos.201200 |bibcode=2020RSOS....701200W}}</ref> and it decreases water loss, which can be crucial for species experiencing long periods out of the water such as [[Biology:Lungfish|lungfish]] species.<ref name=":1">{{cite journal |vauthors=Chew SF, Ong TF, Ho L, Tam WL, Loong AM, Hiong KC, Wong WP, Ip YK |display-authors=6 |title=Urea synthesis in the African lungfish ''Protopterus dolloi''--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure |journal=The Journal of Experimental Biology |volume=206 |issue=Pt 20 |pages=3615–24 |date=October 2003 |pmid=12966053 |doi=10.1242/jeb.00619 |s2cid=9687376|doi-access=free }}</ref> CPS III has been described in [[Biology:Cichlid|cichlid]]s of the ''Alcolapia'' genus,<ref name=":0" /> lungfishes,<ref name=":1" /><ref name=":2">{{cite journal|vauthors=Laberge T, Walsh PJ |title=Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes |journal=Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics |volume=6 |issue=2 |pages=187–94 |date=June 2011 |pmid=21482211 |doi=10.1016/j.cbd.2011.03.001}}</ref><ref>{{cite journal|vauthors=Loong AM, Chng YR, Chew SF, Wong WP, Ip YK |title=Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, ''Protopterus annectens'', during aestivation or exposure to ammonia |journal=Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology |volume=182 |issue=3 |pages=367–79 |date=April 2012 |pmid=22038021 |doi=10.1007/s00360-011-0626-7 |s2cid=6766714}}</ref> the gulf toadfish ''Opsanus beta'',<ref>{{cite journal|vauthors=Kong H, Kahatapitiya N, Kingsley K, Salo WL, Anderson PM, Wang YS, Walsh PJ |title=Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (''Opsanus beta'') |journal=The Journal of Experimental Biology |volume=203 |issue=Pt 2 |pages=311–20 |date=January 2000 |pmid=10607541 |doi=10.1242/jeb.203.2.311|doi-access=free }}</ref> the rainbow trout ''Oncorhyncus mykiss'',<ref>{{cite journal|vauthors=Korte JJ, Salo WL, Cabrera VM, Wright PA, Felskie AK, Anderson PM |title=Expression of carbamoyl-phosphate synthetase III mRNA during the early stages of development and in muscle of adult rainbow trout (''Oncorhynchus mykiss'') |journal=The Journal of Biological Chemistry |volume=272 |issue=10 |pages=6270–7 |date=March 1997 |pmid=9045644 |doi=10.1074/jbc.272.10.6270 |doi-access=free}}</ref><ref>{{cite journal|vauthors=Todgham AE, Anderson PM, Wright PA |title=Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (''Oncorhynchus mykiss'') |journal=Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology |volume=129 |issue=2–3 |pages=527–39 |date=June 2001 |pmid=11423323 |doi=10.1016/S1095-6433(01)00290-2}}</ref> the Atlantic halibut ''Hippoglossus hippoglossus'',<ref>{{cite journal|vauthors=Terjesen BF, Rønnestad I, Norberg B, Anderson PM |title=Detection and basic properties of carbamoyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (''Hippoglossus hippoglossus'' L.) |journal=Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology |volume=126 |issue=4 |pages=521–35 |date=August 2000 |pmid=11026664 |doi=10.1016/S0305-0491(00)00221-2}}</ref> the largemouth bass ''Micropterus salmoides'',<ref>{{cite journal|vauthors=Kong H, Edberg DD, Korte JJ, Salo WL, Wright PA, Anderson PM |title=Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and mRNA in extrahepatic tissues of largemouth bass (''Micropterus salmoides'') |journal=Archives of Biochemistry and Biophysics |volume=350 |issue=2 |pages=157–68 |date=February 1998 |pmid=9473289 |doi=10.1006/abbi.1997.0522}}</ref> the common carp ''Cyprinus carpio'',<ref>{{Cite journal|vauthors=Felskie AK, Anderson PM, Wright PA |date=1998-02-01|title=Expression and Activity of Carbamoyl Phosphate Synthetase III and Ornithine Urea Cycle Enzymes in Various Tissues of Four Fish Species |journal=Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology|volume=119|issue=2|pages=355–364|doi=10.1016/S0305-0491(97)00361-1|issn=1096-4959}}</ref> and in [[Biology:Elasmobranchii|elasmobranchs]] such as the spiny dogfish ''Squalus acanthia''<ref>{{cite journal|vauthors=Hong J, Salo WL, Chen Y, Atkinson BG, Anderson PM |title=The promoter region of the carbamoyl-phosphate synthetase III gene of ''Squalus acanthias'' |journal=Journal of Molecular Evolution |volume=43 |issue=6 |pages=602–9 |date=December 1996 |pmid=8995057 |doi=10.1007/BF02202108 |bibcode=1996JMolE..43..602H |s2cid=6835045}}</ref><ref>{{cite journal|vauthors=Chana-Munoz A, Jendroszek A, Sønnichsen M, Kristiansen R, Jensen JK, Andreasen PA, Bendixen C, Panitz F |display-authors=6 |title=Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (''Squalus acanthias'') provides a molecular tool for biological research and reveals new genes involved in osmoregulation |journal=PLOS ONE |volume=12 |issue=8 |pages=e0182756 |date=2017-08-23 |pmid=28832628 |pmc=5568229 |doi=10.1371/journal.pone.0182756 |bibcode=2017PLoSO..1282756C |doi-access=free}}</ref> for example. This [[Biology:Enzyme|enzyme]] thus seems to be distributed among fish showing different degree of ureotely.

== Reaction pathway ==

=== Ornithine-urea cycle ===
CPS III is a precursor in the [[Chemistry:Urea cycle|ornithine-urea cycle]] (OUC). This pathway occurs in organisms which do not directly excrete ammonia as a catabolic waste. The main function of the OUC is to convert highly toxic nitrogen waste (NH<sub>3</sub>) in urea, which shows less toxicity. This cycle includes five biochemical reactions, the first two of which occur in the [[Biology:Mitochondrion|mitochondrial]] matrix and the three others in the [[Biology:Cytosol|cytosol]]. In fishes, the urea cycle is only found in a few teleosts, mostly air breeders or species living in very specific environments such as alkaline water,<ref>{{cite journal|vauthors=Randall DJ, Wood CM, Perry SF, Bergman H, Maloiy GM, Mommsen TP, Wright PA |title=Urea excretion as a strategy for survival in a fish living in a very alkaline environment |journal=Nature |volume=337 |issue=6203 |pages=165–6 |date=January 1989 |pmid=2911349 |doi=10.1038/337165a0 |bibcode=1989Natur.337..165R |s2cid=4272256}}</ref> and in elasmobranchs.

CPS III is found in the [[Biology:Mitochondrion|mitochondria]] of some [[Biology:Elasmobranchii|elasmobranch]] and in a few teleosts liver and/or extrahepatic tissues. It intervenes in the first reaction of the cycle of the OUC, which is crucial since it limits the rest of the cycle. CPS III thus plays a major role in regulating the amount of ammonia in the cell, by starting its conversion in urea for excretion while maintaining a minimum concentration to maintain [[Biology:Amino acid synthesis|amino acids synthesis]].

=== Carbamoyl phosphate synthesis ===
The reaction catalyzed by CPS III is:<ref>{{cite journal|vauthors=Holden HM, Thoden JB, Raushel FM |title=Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product |journal=Cellular and Molecular Life Sciences |volume=56 |issue=5–6 |pages=507–22 |date=October 1999 |pmid=11212301 |doi=10.1007/s000180050448 |s2cid=23446378}}</ref> 2 ATP + L-glutamine + HCO3- + H20 → 2 ADP + Pi + L-glutamate + carbamoyl phosphat

This reaction occurs in the mitochondrial matrix and include 4 steps:          

# Bicarbonate (HCO<sub>3</sub><sup>−</sup>) is phosphorylated using an [[Chemistry:Adenosine triphosphate|ATP]], generating carboxyphosphate (CHO<sub>6</sub>P<sup>2-</sup>)
# [[Chemistry:Glutamine|Glutamine]] (C<sub>5</sub>H<sub>10</sub>N<sub>2</sub>O<sub>3</sub>) is [[Chemistry:Hydrolysis|hydrolyzed]] into glutamate (C<sub>5</sub>H<sub>9</sub>NO<sub>4</sub>) and [[Chemistry:Ammonia|ammonia]] (NH<sub>3</sub>). 1. and 2. occur concurrently.
# [[Chemistry:Nucleophilic substitution|Nucleophilic substitution]] of the ammonia on carboxyphosphate (substituting the -OH group by a -NH<sub>2</sub> group) generating the intermediate product [[Chemistry:Carbamate|carbamate]] (CH<sub>2</sub>NO<sub>2</sub><sup>−</sup>)
# Nucleophilic substitution of the [[Chemistry:Carbamate|carbamate]] on a second [[Chemistry:Adenosine triphosphate|ATP]], generating the product [[Chemistry:Carbamoyl phosphate|carbamoyl phosphate]] ({{chem|C|H|2|N|O|5|P|2-}}).

CPS III, like CPS I, shows a N-acetylglutamate-dependent, which means that this [[Biology:Effector|allosteric effector]] is required to perform the catalysis.<ref name=":2" />

== Structure ==
CPS III is composed of two subunits: a synthetase and a glutaminase. These two subunits seem to be fused by the N-terminal end of the synthetase<ref name=":3">{{cite journal | vauthors = Hong J, Salo WL, Lusty CJ, Anderson PM | title = Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases | journal = Journal of Molecular Biology | volume = 243 | issue = 1 | pages = 131–40 | date = October 1994 | pmid = 7932737 | doi = 10.1006/jmbi.1994.1638 }}</ref> (Hong et al., 1994)

The 38 first amino acids of the sequence (N-terminal sequence) represent a mitochondrial signal sequence to signal import in the mitochondria.

The glutaminase subunit is located between Phe39 and Ile407 and is itself divided into two domains: an N-terminal domain between Phe39 and Asp165, and a C-terminal glutamine amide transferase domain (GAT) located between Thr166 and Ile407. The cysteine residue Cyst294 along with three histidine residues Hist337, Hist367, and Hist378, have been identified as crucial for the glutamine-dependent activity. In other words, these residues allow CPS III to use glutamine as a substrate.

The synthetase subunit stretches from Lys425 to Gln15032 (C-terminal end) and is also composed of two domains. The first one is located between Lys425 and Ile977 and the second one between Met978 and Gln1502. Each may contain an ATP binding site located between Arg719 and Asp768, and between Arg1260 and Ile1304. It is believed that the C-terminal region contains the binding site for the fixation of the allosteric effector N-acetylglutamate (NAG) which is required for CPS III to function. Two cysteines Cys1328 and Cys1338 have been identified in CPS I, which also use NAG as an allosteric effector, but not in CPS II, which activity is not affected by NAG. Thus, these two cysteine residues appear to probably play a crucial role in the allosteric activity of CPS III.

== Evolution and relationship with CPS I and CPS II ==
CPS III is closer to [[Biology:Carbamoyl phosphate synthetase I|CPS I]] than [[Biology:Carbamoyl phosphate synthase II|CPS II]].<ref name=":3" /> These two enzymes work the same way and use the same allosteric effector. The difference between them is that CPS III uses glutamine as substrate while CPS I use ammonia.

It is believed that these [[Biology:Enzyme|enzyme]]s evolved from each other. One hypothesis is that CPS II appeared first after the fusion of [[Biology:Gene|gene]]s coding for a glutaminase and an ammonia-dependent synthetase. CPS III would then result from the duplication of the glutaminase sequence, creating a second glutamine binding site that evolved into the N-acetylglutamate allosteric site. The last type, CPS I would be the last one to appear after evolving in using ammonia as substrate instead of glutamine.<ref>{{cite journal | vauthors = Devaney MA, Powers-Lee SG | title = Immunological cross-reactivity between carbamyl phosphate synthetases I, II, and III | journal = The Journal of Biological Chemistry | volume = 259 | issue = 2 | pages = 703–6 | date = January 1984 | doi = 10.1016/S0021-9258(17)43514-9 | pmid = 6363405 | doi-access = free }}</ref><ref name=":2" /><ref>{{cite journal | vauthors = Lindley TE, Laberge T, Hall A, Hewett-Emmett D, Walsh PJ, Anderson PM | title = Sequence, expression and evolutionary relationships of carbamoyl phosphate synthetase I in the toad Xenopus laevis | journal = Journal of Experimental Zoology Part A: Ecological Genetics and Physiology | volume = 307 | issue = 3 | pages = 163–75 | date = March 2007 | pmid = 17397070 | doi = 10.1002/jez.a.364 }}</ref>

== References ==
{{Reflist}}

[[Category:EC 6.3.5]]

{{Sourceattribution|Carbamoyl phosphate synthetase III}}