Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

Nguyen, Duy Duc; Pandian, Ramesh; Kim, Doyoun; Ha, Soonhoi; Yoon, Hye‐Jin; Kim, Kap Sun; Yun, Kyung Hee; Kim, Jin-Hahn; Kim, Kyeong Kyu

doi:10.1016/j.bbrc.2014.03.109

Cited by 13 publications

(20 citation statements)

References 23 publications

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“…These enzymes are members of four peptidase families: M17 peptidase (PepB), GAT-1 hydrolase (PepE), isoaspartyl dipeptidase (IadA and IaaA), and M20B peptidase (DapE) families [1–4]. More recently, a microbial M18 peptidase capable of cleaving both Glu and Asp residues from chromogenic substrates was identified and crystallized [5]. …”

Section: Introductionmentioning

confidence: 99%

“…aeruginosa enzyme (PaAP) hydrolyze peptides with N-terminal Asp and Glu residues. Their substrates include dipeptides, tripeptides and larger peptides such as angiotensin I and II [5, 10, 12–16]. However, the human, rodent and rabbit DAPs and the yeast Ape4 inefficiently cleave chromogenic or fluorometric substrates, respectively [12, 13, 16].…”

Section: Introductionmentioning

confidence: 99%

“…falciparum PfM18AAP and P . aeruginosa AP that readily hydrolyze Glu- and Asp-fluorogenic and - p -nitroanilide substrates [5, 11, 17]. …”

Section: Introductionmentioning

confidence: 99%

“…falciparum , bovine, and P . aeruginosa DAPs are dodecameric with tetrahedron-like structures [5, 14, 16, 17, 19]. The X-ray crystal structures have identified the residues that facilitate the coordination of the divalent cations in each subunit, interdigitate with neighboring subunits, line the catalytic pocket, and are required for catalysis [14, 16, 17, 19].…”

Section: Introductionmentioning

confidence: 99%

“…In addition, His to Phe substitutions in the human DAP (DNPEP) showed that eight conserved His residues are important for DAP structure and/or function [13]. Ala substitutions at His401, Asp236 and His82 showed the importance of these residues in coordination of divalent cations and catalysis [5]. …”

Section: Introductionmentioning

confidence: 99%

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Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

et al. 2017

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M18 aspartyl aminopeptidases (DAPs) are well characterized in microbes and animals with likely functions in peptide processing and vesicle trafficking. In contrast, there is a dearth of knowledge on plant aminopeptidases with a preference for proteins and peptides with N-terminal acidic residues. During evolution of the Plantae, there was an expansion and diversification of the M18 DAPs. After divergence of the ancestral green algae from red and glaucophyte algae, a duplication yielded the DAP1 and DAP2 lineages. Subsequently DAP1 genes were lost in chlorophyte algae. A duplication of DAP2-related genes occurred early in green plant evolution. DAP2 genes were retained in land plants and picoeukaryotic algae and lost in green algae. In contrast, DAP2-like genes persisted in picoeukaryotic and green algae, while this lineage was lost in land plants. Consistent with this evolutionary path, Arabidopsis thaliana has two DAP gene lineages (AtDAP1 and AtDAP2). Similar to animal and yeast DAPs, AtDAP1 is localized to the cytosol or vacuole; while AtDAP2 harbors an N-terminal transit peptide and is chloroplast localized. His6-DAP1 and His6-DAP2 expressed in Escherichia coli were enzymatically active and dodecameric with masses exceeding 600 kDa. His6-DAP1 and His6-DAP2 preferentially hydrolyzed Asp-p-nitroanilide and Glu-p-nitroanilide. AtDAPs are highly conserved metallopeptidases activated by MnCl2 and inhibited by ZnCl2 and divalent ion chelators. The protease inhibitor PMSF inhibited and DTT stimulated both His6-DAP1 and His6-DAP2 activities suggesting a role for thiols in the AtDAP catalytic mechanism. The enzymes had distinct pH and temperature optima, as well as distinct kinetic parameters. Both enzymes had high catalytic efficiencies (kcat/Km) exceeding 1.0 x 107 M-1 sec-1. Using established molecular chaperone assays, AtDAP1 and AtDAP2 prevented thermal denaturation. AtDAP1 also prevented protein aggregation and promoted protein refolding. Collectively, these data indicate that plant DAPs have a complex evolutionary history and have evolved new biochemical features that may enable their role in vivo.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…falciparum PfM18AAP and P . aeruginosa AP that readily hydrolyze Glu- and Asp-fluorogenic and - p -nitroanilide substrates [5, 11, 17]. …”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

et al. 2017

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Purification and characterization of a fungal aspartic peptidase from Trichoderma reesei and its application for food and animal feed protein hydrolyses

et al. 2022

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BACKGROUND: Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions. RESULTS: A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent K i -values of 1.75 and 30.2 mg*mL −1 , respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min. CONCLUSION: Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases.

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Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid

Zheng

Tang

et al. 2016

Appl Microbiol Biotechnol

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Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co, with a specific activity of 753.5 U/mg, K value of 24.73 mM, and k /K value of 22.47 mM s. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee ) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

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Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

Cited by 13 publications

References 23 publications

Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

Purification and characterization of a fungal aspartic peptidase from Trichoderma reesei and its application for food and animal feed protein hydrolyses

Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid

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