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Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeonPyrococcus yayanosii CH1
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Huanhuan ZHANG1, 2, Rouke CHEN1, Jun XU1, *
Acta Microbiologica Sinica | 2024, 64(5) : 1494 - 1505
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Acta Microbiologica Sinica | 2024, 64(5): 1494-1505
• Research Articles •
Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeonPyrococcus yayanosii CH1
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Huanhuan ZHANG1, 2, Rouke CHEN1, Jun XU1, *
Affiliations
  • 1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
  • 2 School of Oceanography, Shanghai Jiao Tong University, Shanghai 200240, China
Published: 2024-05-04 doi: 10.13343/j.cnki.wsxb.20230697
Outline
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[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome ofPyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties ofPyprol encoded byPYCH_07700in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeonThermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 ℃ and pH 6.0.Pyprol binding to Co2+ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L.Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidasePfprol fromP.furiosus.Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 ℃ under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase ofP.yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.

hyperthermophilic archaeon  /  Pyrococcus yayanosii  /  prolidase  /  thermostable  /  piezotolerant
Huanhuan ZHANG, Rouke CHEN, Jun XU. Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeonPyrococcus yayanosii CH1[J]. Acta Microbiologica Sinica, 2024 , 64 (5) : 1494 -1505 . DOI: 10.13343/j.cnki.wsxb.20230697
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Prolidase (EC 3.4.13.9) is a peptidase that specifically cleaves the C-terminal proline or hydroxyproline residues of dipeptides. Prolidase has been identified in various organisms to date, including humans[1-2], bacteria[3-4], and archaea[5-7]. In prokaryotes, it is generally believed that prolidase is involved in the proline cycle[8] and bacterial defense against toxins[9]. In humans, prolidase is involved in the degradation of collagen[10]. The activity of prolidase has been found to be abnormally increased in breast cancer[11] and myeloproliferative neoplasms[12]. In addition, the enzyme can also combine with the tumor suppressor p53[13]. So human prolidase is also an important cancer marker. Prolidase has many applications in biotechnology. During cheese fermentation, prolidase can be added to increase the content of proline in the product[14]. In addition, prolidase can be used as an antidote for organophosphorus compounds[15].
Prolidase is a metalloenzyme, and metal ions contribute to stabilizing its structure and anchoring the substrate at the active site[8]. Prolidase always contains two metal-binding cores, and it requires both metal cores to be occupied for full enzymatic activity[16]. The metal-binding site amino acids (Asp-Asp-His-Glu-Glu) are highly conserved[8].
The enzymatic properties of prolidase from hyperthermophilic archaea are different from those of other species. While prolidase from humans andEscherichia coli preferentially bind to Mn2+[4,17], prolidases from hyperthermophilic archaea have the highest activity when binding Co2+[5,7]. It is worth noting that a prolidasePfprol ofPyrococcus furious can also bind Fe2+ under anaerobic conditions[5]. The optimum temperature of prolidases fromP.furious DSM3638 andP. horikoshii OT3 reached 100 ℃[5,7], which is the highest among the prolidases studied. Moreover, prolidases fromPyrococcus species exhibit excellent thermal stability, for example, the prolidase fromP.furiosus DSM 3638 andP.horikoshii OT3 maintained their activity without significant loss when incubated at 100 ℃ for 12 h and 8 h, respectively[5,7].
P.yayanosii CH1 was isolated from the sediment sample collected at a depth of 4 100 m in the Mid-Atlantic Ridge[18]. The optimum growth temperature ofP.yayanosii CH1 is 98 ℃ and the optimum growth pressure is 52 MPa[19]. So in this regard,P.yayanosii CH1 is an obligate piezophilic hyperthermophile and serves as an important model organism for studying the mechanisms of high-pressure adaptation in microorganisms[20]. A putative prolidase encoding genePYCH_07700 was found in the genome ofP.yayanosii CH1. This study will present the results of the characterization of the enzymatic properties of the above-mentioned prolidase, namelyPyprol.Pyprol was obtained by heterologous overexpression inThermococcus kodakarensis TS559, which is an agmatine auxotroph strain[21-22]. The effect of hydrostatic pressure on the enzymatic activity ofPyprol was investigated, which provides clues for expanding the application of this type of prolidase.
1 Materials and Methods
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1.1 Strains, plasmids, and culture conditions
The strains and plasmids utilized in this study are outlined inTable 1.E.coli DH5α was cultured in Luria-Bertani (LB) medium at 37 ℃.P.yayanosii A1 was cultivated under anaerobic at 95 ℃ in TRM medium[23].T.kodakarensis TS559 strains were cultivated in the artificial seawater (ASW-YT) liquid medium supplemented with 1 mmol/L agmatine under anaerobic at 85 ℃.
1.2 Bioinformatics analysis
The amino acid sequence of the experimentally characterized prolidasePfprol (WP_011012489.1) ofP.furiosus was used as a query to BLAST against the genome sequence ofP.yayanosii CH1 (GenBank accession number: NC_015680.1). Putative prolidases includingPyprol (WP_013905514.1),Pfprol, and other homologous sequences were retrieved from GenBank. The amino acid sequences of obtained prolidases were aligned using ClustalX2 and visualized using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). The phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA (version 7)[24]. Bootstrap analysis was computed with 1 000 replicates.
1.3 Cloning of prolidase encoding genes
Using the genomic DNA ofP.yayanosii A1 as a template, the full-length sequence of genePYCH_07700 was amplified using primer 0770-kod-F/R (Table 2). Plasmid pTE1 was used as the template DNA and the primer pTE1-F/R (Table 2) was used in PCR to amplify the backbone of thisE.coli-T.kodakarensis shuttle vector[25]. Using ClonExpress Ⅱ One Step Cloning Kit (Vazyme, China), genePYCH_07700 was ligated to the pTE1 plasmid and transformed into DH5α. The prolidase encoding genePF_1343 ofP.furiosus DSM3638 was cloned into pTE1 using same strategy with changes in PCR primers (1343-kod-F/R) and corresponding template DNA (Table 2). In order to purify the protein, a 12×His tag was added to the C-terminus ofPYCH_07700 andPF_1343, respectively. Plasmid constructed with correct insertion of either genePYCH_07700 orPF_1343 was confirmed by colony PCR and DNA-sequencing analysis.
Genetic manipulations ofT.kodakarensis were carried out under anaerobic conditions. The transformation ofT.kodakaraensis was performed as previously described[25]. The host strainT.kodakarensis TS559 was cultivated in ASW-YT liquid medium supplemented with 1 mmol/L agmatine at 85 ℃ for 10 h, and the cells were harvested by centrifugation (6 500×g, 5 min). The harvested cells were resuspended in 200 μL of 0.1 mol/L CaCl2 and kept on ice for 30 min. Then, 3 μg of plasmid was added to the cell suspension and incubated on ice for 1 h, followed by a heat shock at 85 ℃ for 45 s and incubation on ice for 10 min. The cell suspension was added to 5 mL ASW-YT liquid medium and incubated at 85 ℃ for 4 h. The culture was spread onto ASW-YT solid medium without agmatine and cultured at 85 ℃ until colonies were observed. The positive colonies were confirmed by colony PCR and DNA sequencing analysis.
1.4 Prolidase expression and purification inThermococcus kodakarensis
The recombinant strains were inoculated into ASW-YT medium and cultured at 85 ℃ for 15 h under anaerobic conditions. Cells were collected by centrifugation at 10 000×g for 5 min at room temperature. The cells were resuspended in 50 mmol/L Tris-HCl (pH 8.0) containing 0.5 mol/L NaCl and then crushed by sonication on ice. The supernatant was collected by centrifugation at 10 000×g for 30 min at 4 ℃. Proteins were purified using Ni-NTA 6FF Sefinose Resin Kit (Sangon, China). Imidazole in the buffer that was used to elute the overexpressed protein was removed using Millipore 10 kDa ultrafiltration tubes. The purified protein was finally stored in 50 mmol/L Tris-HCl (pH 8.0) containing 0.5 mol/L NaCl.
1.5 Prolidase activity assay
Prolidase activity was determined as previously described[5]. The 50 μL reaction mixture contained 50 mmol/L MOPS buffer (pH 7.0), 200 mmol/L NaCl, 5% glycerol, 0.1 mg/mL BSA protein, and 1.2 mmol/L CoCl2. After adding an appropriate amount of protein, react at 100 ℃ for 5 min to allow the protein to bind to the metal ion. Add Met-Pro at a final concentration of 10 mmol/L and react at 100 ℃ for 10 min. Add 50 μL of acetic acid to stop the reaction, then add 50 μL of 3% (W/V) ninhydrin solution to react at 100 ℃ for 10 min. After cooling to room temperature, use a microplate reader to measure the absorbance at 515 nm. The activity unit of prolidase is defined as the amount of enzyme that releases one micromole of proline per minute.
1.6 Influence of temperature, pH, and metal preference on enzyme activity
The optimum temperature ofPyprol was determined in the range of 40–100 ℃. The optimum pH ofPyprol was determined in the range of pH 4.0–8.0. The buffer used were 50 mmol/L of CH3COOH-CH3COONa (pH 4.0–5.0), NaH2PO4-Na2HPO4 (pH 6.0–7.0), Tris-HCl (pH 8.0). To determine the optimal metal ion ofPyprol,Pyprol was combined with Co2+, Mn2+, Zn2+, Ca2+, Cu2+, Ni2+, and Mg2+, respectively, and then reacted with the substrate. To evaluate the effect of metal ion concentration on the enzyme activity,Pyprol was combined with Co2+ at a final concentration of 0–6 mmol/L and then reacted with the substrate.
1.7 Influence of hydrostatic pressure on enzyme activity
To assess the effect of hydrostatic pressure onPyprol activity, prolidase activity assays were performed at 0.1, 10, 20, 30, 40, and 52 MPa at 100 ℃, respectively. To evaluate the effect of high hydrostatic pressure on prolidase activity at different temperatures, the prolidase activity under its optimum hydrostatic pressure was measured at 40, 70, and 100 ℃, respectively. High pressure was achieved and controlled by adding water through the hand-operated pump that was equipped with a pressure gauge[26-27]. The pin-closure pressure vessels were used in this study (constructed by Nantong Feiyu Petroleum Technology Development Co., Ltd., China).
1.8 Influence of temperature and hydrostatic pressure on enzyme stability
The thermal stability ofPyprol was determined by incubating the assays at specific temperatures (80, 90, and 100 ℃) for different periods (1, 2, 3, 4, and 5 h), and measuring the residual activity. The high hydrostatic pressure stability ofPyprol was determined by incubating at 80 ℃ under 20 MPa and 40 MPa for 1 h, and the residual enzyme activity was determined.
1.9 Determination of enzyme kinetics constants
The enzyme reaction rate ofPyprol was determined in the Met-Pro concentration range of 1–10 mmol/L at 0.1 MPa and 40 MPa, respectively. The reaction was conducted under the condition of 50 mmol/L NaH2PO4-Na2HPO4 buffer (pH 6.0), 1.2 mmol/L CoCl2, and 100 ℃. The results were fitted using the Michaelis-Menten equation in the data analysis and graphing software Origin.
2 Results
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2.1 Overexpression and purification of a prolidasePyprol fromPyrococcus yayanosii inThermococcus kodakarensis
InP.yayanosii CH1, the proteinPyprol encoded byPYCH_07700 is predicted to function as a prolidase. InPyprol, there are two conserved structural domains, namely creatinase_N located at the N-terminus, and peptidase_M24 situated at the C-terminus (Figure S1, data was deposited in the China National Microbiology Data Center, accession No.: NMDCX0000258). BLASTp analysis showed thatPyprol exhibited high similarity to prolidases fromP.furiosus DSM3638 andP.horikoshii OT3, with amino acid sequence similarities of 76% and 75%, respectively (Figure 1). Pyprol also exhibits a high structural similarity to the prolidase fromP.furiosus (Figure S2, data was deposited in the China National Microbiology Data Center, accession No.: NMDCX0000259). Multiple alignments ofPyprol with its homologous proteins revealed thatPyprol contained the conserved metal-binding sites (Asp-Asp-His-Glu-Glu). These results suggested thatPyprol was a putative prolidase.
To overexpress the prolidase fromP.yayanosii CH1 inT.kodakarensis TS559,PYCH_07700 gene fragment was cloned into a shuttle vector plasmid pTE1 in the downstream region of the glutamate dehydrogenase (Pgdh) promoter fromP.furiosus DSM3638 (Figure 2A). The recombinant strains containing plasmids pTE-Pyprol and pTE-Pfprol were cultured in ASW-YT medium at 85 ℃ for 15 h, and cells were harvested by centrifugation. The cells were crushed by sonication, and the supernatants were collected by centrifugation. The prolidases were purified by using nickel-charged resin. The purified proteins were analyzed using SDS-PAGE (Figure 2B), and the results showed that the molecular weight of the recombinant protein was approximately 43 kDa which was consistent with the theoretical relative molecular weight.
2.2 Optimal temperature, optimal pH, and metal ion preference ofPyprol
The activity ofPyprol was determined by measuring its ability to cleave the Met-Pro dipeptide substrate over a temperature range of 40–100 ℃ (Figure 3A). The results indicated that the optimal temperature forPyprol activity was 100 ℃, and there was a notable decrease in activity when the temperature dropped below 70 ℃. The activity ofPyprol was determined over a pH range of 4.0–8.0 (Figure 3B). The optimal pH forPyprol activity was found to be 6.0, and a significant decline in activity was observed at pH 4.0. WhenPyprol was bound to different metal ions (Figure 3C), significant variations in activity were observed.Pyprol exhibited the highest activity when bound to Co2+. It also retained 93% of the activity observed when bound to Co2+ when it was bound to Mn2+. However, the binding of Zn2+, Ca2+, Cu2+, Ni2+, and Mg2+ resulted in almost undetectable prolidase activity. The results demonstrated thatPyprol exhibited maximum activity when bound to 1.2 mmol/L Co2+, but even when bound to 0.6 mmol/L Co2+, it retained 81% of the maximum activity (Figure 3C). Additionally, we observed thatPyprol retained 29% activity even in the absence of additional metal ions, compared to that of when 1.2 mmol/L Co2+ was added. This may be attributed to the binding of trace amounts of Co2+ from the culture medium toPyprol.
2.3 Effect of hydrostatic pressure onPyprol activity
The activity ofPyprol was found to be higher under high hydrostatic pressure compared to atmospheric pressure. At 40 MPa,Pyprol exhibited the highest activity (Figure 3D). Furthermore, the extent of enhancement inPyprol activity varied at different temperatures under high hydrostatic pressure. At 40, 70, and 100 ℃, compared to 0.1 MPa,Pyprol activity increased by 67%, 31%, and 24%, respectively (Table 3). This indicated that high hydrostatic pressure has a significant impact onPyprol activity at lower temperatures, especially at 40 ℃. Notably, at 40 ℃ and 40 MPa, the specific activity ofPyprol reached 967 U/mg, which is close to the specific activity observed at 70 ℃ and 0.1 MPa (1 120 U/mg). Similarly, we observed that the activity ofPfprol was higher at 20 MPa compared to atmospheric pressure. Additionally, compared to 0.1 MPa, at 20 MPa, the enzyme activity ofPfprol at 40, 70, and 100 ℃ increased by 47%, 27%, and 24%, respectively.
2.4 Kinetic constants
We used Met-Pro as the substrate and determined the kinetic constants ofPyprol at 0.1 MPa and 40 MPa. The results revealed that theKm values ofPyprol at 0.1 MPa and 40 MPa were similar, but at 40 MPa,Pyprol exhibited a higherVmax value compared to that at 0.1 MPa (Table 4). Thekcat/Km value ofPyprol at 40 MPa was 1.16 times higher than that at 0.1 MPa.
2.5 Thermal stability and high hydrostatic pressure stability
Pyprol was incubated under different temperatures and hydrostatic pressure conditions, followed by the measurement of prolidase activity. The results (Figure 4A) showed that after incubatingPyprol at 80 ℃ and 90 ℃ for 5 h, it retained 64% and 55% of its activity, respectively. However, after incubatingPyprol at 100 ℃ for 1 h, its activity decreased to only 41% (Figure 4A). On the other hand, after incubatingPyprol at 20 MPa and 40 MPa for 1 h, it still retained 88% and 78% of its activity, respectively (Figure 4B).
3 Discussion
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In this study, we reported the characterization of a prolidasePyprol fromP.yayanosii CH1. To the best of our knowledge, this is the first report about a prolidase that exhibits higher activity under high hydrostatic pressure than under atmospheric pressure.
Previous studies have found that the native prolidase (N-prol) purified fromP.furiosus and the recombinant prolidase (R-prol) expressed inE.coli showed no significant differences in enzymatic properties[5]. However, when Met-Pro was used as the substrate, thekcat/Km value of R-prol was 1.64 times higher than that of N-prol[5]. To avoid the host background effect, we chose hyperthermophilic archaeonT.kodakarensis, which is a close relative ofP.yayanosii CH1, as the surrogate host to overexpress the prolidasePyprol[22]. As shown in a previous report, successful expression of the soluble hydrogenase I (SHⅠ) fromP.furiosus has been achieved inT.kodakarensis TS559[25]. We anticipated that the prolidase obtained fromT.kodakarensis TS559 may exhibit enzymatic properties more similar to the native prolidase fromP.yayanosii.
For comparison, we also cloned the prolidase encoding gene (PF_1343) ofP.furiosus and overexpressed it inT.kodakarensis TS559 (Figure 2B). For bothPyprol andPfprol, the prolidase activity was higher under high hydrostatic pressure than at of under atmospheric pressure (Table 3). At 40 ℃ and 0.1 MPa, the specific enzyme activity ofPyprol (578 U/mg) is 15.21 fold of that ofPfprol (38 U/mg). This indicated that at 40 ℃,Pyprol has greater potential in practical applications.
A phylogenetic analysis ofPyprol indicates that prolidase from diverse hyperthermophiles, including that from three genus ofThermococcaceae,Sulfolobus,Archaeoglobus andThermotoga are located within the same branch (Figure 5). It is interesting that the prolidase fromT.barophilus MP, which was the first true hyperthermophilic piezophilic archaeon isolated[28], showed close relationship with prolidases from thePyrococcus genus. It is commonly believed that enzyme activity decreases under high hydrostatic pressure[29]. However, there were reports that proteases derived fromMethanocaldococcus jannaschii exhibited activity 3.4 times higher at 50 MPa 125 ℃ compared to 10 MPa 125 ℃[30]. As well as that, we found that the activity ofPyprol at high hydrostatic pressure was significantly higher than that at atmospheric pressure. Moreover, we observed thatPfprol exhibits a 25% increase in enzyme activity at 20 MPa compared to 0.1 MPa (Table 3). It is indicated that the prolidase from a deep-sea microbe may have a potential similarity in their enzymatic properties.
P.yayanosii CH1 lacks a complete pathway for synthesizing proline[31], indicating the need to acquire proline from the external environment. The unique cyclic structure of proline makes the peptide bonds surrounding the proline residue resistant to degradation, and prolidase is one of the few enzymes in organisms capable of degrading proline residues[32]. The higher activity ofPyprol under high hydrostatic pressure compared to atmospheric pressure suggests its potential role in proline acquisition forP.yayanosii CH1 under high hydrostatic pressure conditions. We deletedPYCH_07700 inP.yayanosii, but no significant decrease in biomass was observed under high hydrostatic pressure (result not shown). Whether functional compensation by other enzymes capable of degrading proline residues inP.yayanosii exist or not will be the focus of future experiments.
Funding
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  • National Key Research and Development Program of China(2020YFA0906800)
  • National Natural Science Foundation of China (General Program)(41976085)
  • 国家自然科学基金面上项目(42276091)
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doi: 10.13343/j.cnki.wsxb.20230697
  • Receive Date：2023-11-14
  • Online Date：2026-03-19
  • Published：2024-05-04
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History
  • Received：2023-11-14
  • Accepted：2024-02-19
Funding
National Key Research and Development Program of China(2020YFA0906800)
National Natural Science Foundation of China (General Program)(41976085)
国家自然科学基金面上项目(42276091)
Affiliations
    1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
    2 School of Oceanography, Shanghai Jiao Tong University, Shanghai 200240, China

Corresponding:

*XU Jun, E-mail:
References
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https://castjournals.cast.org.cn/joweb/wswxb/EN/10.13343/j.cnki.wsxb.20230697
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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