Biology:Omega-amidase
| Omega-amidase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 A 3D cartoon depiction of the crystal structure of mouse nitrilase 2. | |||||||||
| Identifiers | |||||||||
| EC number | 3.5.1.3 | ||||||||
| CAS number | 9025-19-8 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
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In enzymology, an omega-amidase (EC 3.5.1.3) is an enzyme that catalyzes the chemical reaction
- a monoamide of a dicarboxylic acid + H2O [math]\displaystyle{ \rightleftharpoons }[/math] a dicarboxylate + NH3
Thus, the two substrates of this enzyme are monoamide of a dicarboxylic acid and H2O, whereas its two products are dicarboxylate and NH3.
This enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides. The systematic name of this enzyme class is omega-amidodicarboxylate amidohydrolase. This enzyme is also called alpha-keto acid-omega-amidase. This enzyme participates in glutamate metabolism and alanine and aspartate metabolism. This enzyme can be found in mammals, plants, and bacteria.[1]
Structure and active site
Omega-amidase has two independent monomers that have structure organizations similar to other nitrilase enzymes found in bacteria.[2] Each monomer has a four layered alpha/beta/beta/alpha conformation.[2] The enzyme is asymmetrical and contains a carbon-nitrogen hydrolase fold.[2]
Just as omega-amidase shares a general structure organization as other nitrilases, omega-amidase also contains the same catalytic triad within the active site. This triad of residues includes a nucleophilic cysteine, a glutamate base, and a lysine, all of which are conserved within the structure.[2] In addition to the catalytic triad, omega-amidase also contains a second glutamate that assists in substrate positioning.[3] This second glutamate is why omega-amidase has no activity with glutamine or asparagine, even though they are sized similarly to typical substrates.[4]
Mechanism
Omega amidase catalyzes the deamidation of several different alpha-keto acids into ammonia and metabolically useful carboxylic acids[5] The general mechanism is the same as for other nitrilases: binding of the substrate to the active site, followed by release of ammonia, formation of a thioester intermediate at the cysteine, binding of water and then release of the carboxylic acid product.[3] Specifically, the active site cysteine acts as a nucleophile and binds to the substrate.[6] The catalytic triad glutamate transfers a proton to the amide group to create and release ammonia.[7] The remaining thioester intermediate is stabilized by the lysine and the backbone amino group following the cysteine.[6] This intermediate is attacked by water to form a stable tetrahedral intermediate.[7] This intermediate breaks down to release the carboxylic acid and restore the enzyme.[7]
Biology
Omega-amidase operates in coordination with glutamine transaminase to finish off the methionine salvage cycle in bacteria and plants.[1] In the last step to obtain methionine from α-ketomethylthiobutyrate(KMTB), glutamine transaminase K(GTK) converts glutamine to α-ketoglutaramate(KGM).[1] KGM is the main substrate for omega amidase, but KGM exists mainly in the ring form at physiological conditions.[4] Omega-amidase has a higher affinity for the open linear form of KGM that forms more readily at pH 8.5.[8] GTK catalyzes a reversible reaction, but coupling it with omega-amidase makes the transamination reaction irreversible at physiological conditions.[8]
Due to omega-amidase's ability to convert toxic substrates like KGM into components that can be used by other processes, this enzyme can be considered a repair enzyme.[9] Some such substrates are linked to diseases or conditions such as hyperammonemia.[10] A list of some of the substrates that omega-amidase catalyzes may be found in Table 1.
| Substrate | Product |
|---|---|
| α-Ketoglutaramate | α-Ketoglutarate |
| α-Ketosuccinamate | Oxaloacetate |
| L-2-Hydroxysuccinamate | L-Malate |
| Succinamic Acid | Succinylmonohydroxamic Acid[11] |
| Glutaramic Acid | Glutarylmonohyoxamic Acid[11] |
Medical relevance
The NIT2 gene in humans has been found to be identical to omega-amidase.[9] The gene has the highest expression in the liver and kidney, but is also expressed in almost every human tissue.[5] Overexpression of the NIT2 gene results in decreasing cell proliferation and growth in HeLa cells, which indicates that the gene may have a role in tumor suppression.[9] However further studies are necessary to determine the effect on specific cancers, as a study done with colon cancer cells showed that downregulation of NIT2 induced cell cycle arrest.[12] In addition to tumor suppression, NIT2/omega-amidase may be useful for detection and conversion of oncometabolites.[13] Because omega-amidase is able to control concentration of toxic substrates such as KGM, it is likely that NIT2 can serve the same purpose.[13]
References
- ↑ 1.0 1.1 1.2 "Evidence that glutamine transaminase and omega-amidase potentially act in tandem to close the methionine salvage cycle in bacteria and plants". Phytochemistry 113: 160–9. May 2015. doi:10.1016/j.phytochem.2014.04.012. PMID 24837359. Bibcode: 2015PChem.113..160E.
- ↑ 2.0 2.1 2.2 2.3 "Functional proteomic and structural insights into molecular recognition in the nitrilase family enzymes". Biochemistry 47 (51): 13514–23. December 2008. doi:10.1021/bi801786y. PMID 19053248.
- ↑ 3.0 3.1 3.2 "The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning". The Journal of Biological Chemistry 288 (40): 28514–23. October 2013. doi:10.1074/jbc.m113.503284. PMID 23946488.
- ↑ 4.0 4.1 "Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation". The Journal of Biological Chemistry 287 (31): 25715–26. July 2012. doi:10.1074/jbc.m111.259119. PMID 22674578.
- ↑ 5.0 5.1 "Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine". Analytical Biochemistry 391 (2): 144–50. August 2009. doi:10.1016/j.ab.2009.05.025. PMID 19464248.
- ↑ 6.0 6.1 "Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy". FEBS Letters 277 (1–2): 112–4. December 1990. doi:10.1016/0014-5793(90)80821-y. PMID 2269339.
- ↑ 7.0 7.1 7.2 "Microbial nitrilases: versatile, spiral forming, industrial enzymes". Journal of Applied Microbiology 106 (3): 703–27. March 2009. doi:10.1111/j.1365-2672.2008.03941.x. PMID 19040702.
- ↑ 8.0 8.1 "Identification and characterization of omega-amidase as an enzyme metabolically linked to asparagine transamination in Arabidopsis". Phytochemistry 99: 36–43. March 2014. doi:10.1016/j.phytochem.2013.12.020. PMID 24461228. Bibcode: 2014PChem..99...36Z.
- ↑ 9.0 9.1 9.2 "Hits, Fhits and Nits: beyond enzymatic function". Advances in Enzyme Regulation 51 (1): 208–17. 2011. doi:10.1016/j.advenzreg.2010.09.003. PMID 21035495.
- ↑ "New recombinant producer of human ω-amidase based on Escherichia coli". Applied Biochemistry and Microbiology 53 (3): 290–295. May 2017. doi:10.1134/s0003683817030115.
- ↑ 11.0 11.1 "Hydrolysis and transfer reactions catalyzed by omega-amidase preparations". The Journal of Biological Chemistry 215 (1): 441–60. July 1955. doi:10.1016/S0021-9258(18)66051-X. PMID 14392177.
- ↑ "Downregulation of NIT2 inhibits colon cancer cell proliferation and induces cell cycle arrest through the caspase-3 and PARP pathways". International Journal of Molecular Medicine 35 (5): 1317–22. May 2015. doi:10.3892/ijmm.2015.2125. PMID 25738796.
- ↑ 13.0 13.1 "The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites". Biology 6 (2): 24. March 2017. doi:10.3390/biology6020024. PMID 28358347.
Further reading
- "Enzymatic synthesis of L-pipecolic acid and L-proline". The Journal of Biological Chemistry 229 (2): 789–800. December 1957. doi:10.1016/S0021-9258(19)63684-7. PMID 13502341.
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