N-terminal nucleophile hydrolases
| N-terminal nucleophile hydrolases | |
|---|---|
 HUMAN ASPARTYLGLUCOSAMINIDASE[1] | |
| Identifiers | |
| Symbol | Ntn-hydrolases |
| Pfam clan | CL0052 |
| ECOD | 210.1 |
In molecular biology, the N-terminal nucleophile (Ntn)-hydrolases are a structural superfamily of evolutionarily related enzymes that have diverged beyond any recognisable sequence similarity.[2]
Structure
Ntn-hydrolases share a characteristic "αββα-fold" - a four-layered structure with two antiparallel β-sheets sandwiched between α-helical layers. However, the packing angles between the β-sheets vary significantly (5-35°) across different enzymes.
Despite minimal sequence similarity, the researchers identified eight completely conserved secondary structural elements (termed "region C") that are essential for the fold. Five of these elements (β4, β5, β11, β12 strands and α11 helix) contain most of the functionally important residues.[3]
Catalytic mechanism
All enzymes use a similar catalytic strategy with:
- An N-terminal nucleophile (threonine, serine, or cysteine) that acts as both nucleophile and catalytic base
- Formation of a covalent intermediate during substrate hydrolysis
- An oxyanion hole that stabilises the reaction intermediate
While the core catalytic machinery is conserved, the substrate binding sites and some aspects of the oxyanion hole differ between enzymes, reflecting their different substrate specificities.[3]
Examples of proteins belonging to this superfamily
Human Subfamilies
1. Class II Glutamine Amidotransferases (GAT)
These enzymes use cysteine nucleophiles and release ammonia from glutamine for biosynthetic reactions. Deficiencies cause severe diseases (e.g., asparagine synthetase deficiency, congenital myasthenic syndrome). Some examples are ASNS, GPAT and GFAT1/2.
2. PVA Subfamily (Lysosomal Hydrolases)
These proteins use cysteine nucleophiles and hydrolyse fatty acid-amide bonds in sphingolipids. Deficiencies lead to lysosomal storage diseases like Farber disease. Examples are acid ceramidase (ASAH1), NAAA, PLBD1/2, and secernins,
3. Proteasome Subunits
They use threonine nucleophiles and form the catalytic core of the 20S proteasome. Three active β-subunits (β1, β2, β5) degrade ubiquitinated proteins. Immunoproteasome variants (β1i, β2i, β5i) are induced by interferon-γ,
4. Asparaginases (AGA Family)
Use threonine nucleophiles. This group includes lysosomal aspartylglucosaminidase (AGA), ASRGL1, and TASP1. AGA deficiency causes aspartylglucosaminuria, a lysosomal storage disorder, while ASRGL1 has potential as cancer therapeutic.
5. γ-Glutamyl Transpeptidases (GGT)
The six or seven members (GGT1-7) of this group use threonine nucleophiles and transfer or hydrolyse γ-glutamyl groups from glutathione and other substrates. GGT1 is a diagnostic marker for liver disease; its deficiency causes glutathionuria.
Bacterial members
Bacteria possess multiple Ntn-hydrolases including penicillin G/V acylases (e.g., from E. coli, Bacillus sphaericus, and Streptomyces mobaraensis), γ-glutamyl transpeptidases, isoaspartyl dipeptidases, and specialised enzymes such as N-acyl homoserine lactone acylases (e.g., PvdQ from Pseudomonas aeruginosa). Some Ntn-hydrolases such as bile salt hydrolases are widespread in probiotic lactic acid bacteria and play roles in bile detoxification and gut colonisation.[6][7] In addition, unusual variants like β-aminopeptidases (e.g., BapA, DmpA-like family) and peptide amidases are also found in bacteria, often with unique substrate specificities.[8]
Examples in yeast
Yeast possesses several major Ntn-hydrolases, which play crucial roles in protein degradation and metabolic processes. The most prominent examples include subunits of the 20S proteasome and gamma-glutamyl transpeptidase.[9][10] As an example, the 20S proteasome in Saccharomyces cerevisiae has multiple β-type subunits (such as PRE2, PRE3, PRE4) that are classical Ntn-hydrolases activated by autocatalytic cleavage to reveal the N-terminal threonine nucleophile.[11] Gamma-Glutamyl Transpeptidase (ECM38) regulates glutathione metabolism, cellular redox status, and detoxification processes.[12]
Clinical relevance
Many Ntn-hydrolases are clinically important as disease-associated proteins, diagnostic markers, or therapeutic targets.[5]
References
- ↑ Oinonen C, Tikkanen R, Rouvinen J, Peltonen L (December 1995). "Three-dimensional structure of human lysosomal aspartylglucosaminidase". Nature Structural Biology. 2 (12): 1102–1108. doi:10.1038/nsb1295-1102. PMID 8846222.
- ↑ Brannigan JA, Dodson G, Duggleby HJ, Moody PC, Smith JL, Tomchick DR, et al. (November 1995). "A protein catalytic framework with an N-terminal nucleophile is capable of self-activation". Nature. 378 (6555): 416–419. Bibcode:1995Natur.378..416B. doi:10.1038/378416a0. PMID 7477383.
- 1 2 Oinonen C, Rouvinen J (December 2000). "Structural comparison of Ntn-hydrolases". Protein Science. 9 (12): 2329–2337. doi:10.1110/ps.9.12.2329. PMC 2144523. PMID 11206054.
- ↑ Lodola A, Branduardi D, De Vivo M, Capoferri L, Mor M, Piomelli D, et al. (28 February 2012). "A catalytic mechanism for cysteine N-terminal nucleophile hydrolases, as revealed by free energy simulations". PLOS ONE. 7 (2) e32397. Bibcode:2012PLoSO...732397L. doi:10.1371/journal.pone.0032397. PMC 3289653. PMID 22389698.
- 1 2 Linhorst A, Lübke T (May 2022). "The Human Ntn-Hydrolase Superfamily: Structure, Functions and Perspectives". Cells. 11 (10): 1592. doi:10.3390/cells11101592. PMC 9140057. PMID 35626629.
- ↑ Bokhove M, Nadal Jimenez P, Quax WJ, Dijkstra BW (January 2010). "The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket". Proceedings of the National Academy of Sciences of the United States of America. 107 (2): 686–691. Bibcode:2010PNAS..107..686B. doi:10.1073/pnas.0911839107. PMC 2818923. PMID 20080736.
- ↑ Kusada H, Arita M, Tohno M, Tamaki H (6 June 2022). "Bile Salt Hydrolase Degrades β-Lactam Antibiotics and Confers Antibiotic Resistance on Lactobacillus paragasseri". Frontiers in Microbiology. 13 858263. doi:10.3389/fmicb.2022.858263. PMC 9207391. PMID 35733973.
- ↑ Merz T, Heck T, Geueke B, Mittl PR, Briand C, Seebach D, et al. (November 2012). "Autoproteolytic and catalytic mechanisms for the β-aminopeptidase BapA--a member of the Ntn hydrolase family". Structure. 20 (11): 1850–1860. doi:10.1016/j.str.2012.07.017. PMID 22980995.
- ↑ Huber EM, Heinemeyer W, Li X, Arendt CS, Hochstrasser M, Groll M (March 2016). "A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome". Nature Communications. 7 (1) 10900. Bibcode:2016NatCo...710900H. doi:10.1038/ncomms10900. PMC 4792962. PMID 26964885.
- ↑ Mehdi K, Thierie J, Penninckx MJ (November 2001). "gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione". The Biochemical Journal. 359 (Pt 3): 631–637. doi:10.1042/0264-6021:3590631. PMC 1222185. PMID 11672438.
- ↑ Arendt CS, Hochstrasser M (July 1997). "Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation". Proceedings of the National Academy of Sciences of the United States of America. 94 (14): 7156–7161. Bibcode:1997PNAS...94.7156A. doi:10.1073/pnas.94.14.7156. PMC 23776. PMID 9207060.
- ↑ Mitrić A, Castellano I (November 2023). "Targeting gamma-glutamyl transpeptidase: A pleiotropic enzyme involved in glutathione metabolism and in the control of redox homeostasis". Free Radical Biology & Medicine. 208: 672–683. doi:10.1016/j.freeradbiomed.2023.09.020. PMID 37739139.