ABSTRACT
S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.
The addition of external NAD+ stabilizes the structure of P. horikoshii SAHH and enhances its enzyme activity.
The use of enzymes has attracted attention from researchers because enzymes, as biocatalysts, have high reaction selectivity and can promote chemical reactions under mild conditions, aiding their applicability in the field of green chemistry. Since industrial application of enzymes requires extreme conditions such as, high temperature, tolerance to surfactants or organic solvents, and long-term storage, there is a need to improve enzyme stability. Microorganisms that grow under extreme conditions such as high temperature and pressure and non-neutral pH produce thermostable enzymes (Toplak et al.2013). In particular, thermostable enzymes have several advantages, such as ease of purification, increased reactivity, exceptional stability, higher process yield, and lower contamination risk (Mozhaev 1993). In addition, these enzymes are often resistant to oxidants, denaturants, and surfactants (Mozhaev 1993; Toplak et al.2013).
Pyrococcus horikoshii OT3, an anaerobic archaeon, was originally discovered in a hydrothermal vent in the Okinawa Trough in the Pacific Ocean. As a hyperthermophile, this microorganism exhibits optimal growth conditions at 98 °C (González et al.1998). The molecular properties of many enzymes in P. horikoshii have been investigated (Ando et al.2002; Yabuta et al.2015; Goda et al.2018; Kawakami et al.2018). Thus, P. horikoshii has been recognized as a good genetic resource for producing thermostable enzymes.
S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important intermediates in methionine metabolism (Figure 1). SAM acts as a major methyl donor and is involved in various methylation reactions in vivo, such as DNA and histone methylation (Ye et al.2017). An estimated 0.6%-1.6% of genes in various organisms, including human, mouse, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, yeast, and Escherichia coli encode a SAM-dependent methyltransferase (Katz et al.2003). In transmethylation reactions, SAM is converted to SAH, a potent inhibitor of methyltransferases (Shatrov et al.1999; Halsted et al.2011; DeArmond et al.2017). Therefore, the ratio of SAM to SAH is an indicator of the methylation capacity.
Methionine cycles. Ado, adenosine; Hcy, L-homocysteine; Met, methionine; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine; 1, SAM-dependent methyltransferase; 2, S-adenosyl-L-homocysteine hydrolase (SAHH); 3, methionine synthase; and 4, S-adenosyl-L-methionine synthase.
Liquid chromatography-mass spectrometry (LC-MS) methods using stable isotope-labeled SAM and SAH have been developed (Stabler and Allen 2004). However, labeled SAH is not commercially available. Therefore, SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of L-homocysteine (Hcy) and adenosine (Ado) to SAH, is used to produce labeled SAH (Stabler and Allen 2004). Enzymatic production of SAH is particularly effective using thermostable SAHH.
The enzymatic properties of several thermostable SAHH have been reported (Porcelli et al.2000; Porcelliet et al.2005; Lozada-Ramírez et al.2013; Qian et al.2014). To increase the yield of SAH, we searched for the genes encoding SAHH in the P. horikoshii genome, produced recombinant enzymes using the E. coli system, and investigated their enzymatic properties.
Materials and methods
Chemicals
Adenosine was purchased from Nacalai Tesque Inc. (Kyoto, Japan). DL-Homocysteine was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). SAH was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). All other chemicals were of analytical grade and were procured from commercial sources.
Phylogenetic analysis
To conduct phylogenetic analysis, maximum likelihood phylogenetic trees were constructed using the Jones-Taylor-Thornton (JTT) model in MEGA X (Kumar et al.2018). Statistical support was obtained using 1000 bootstrap replicates.
Expression and purification of recombinant P. horikoshii SAHH
P. horikoshii genomic DNA was a gift from Prof. Sakuraba (Kagawa University). The open reading frame of PH0540 was amplified from the genomic DNA of P. horikoshii using the following primers: PH0540-F (5′-CGCGCGGCAGCCATATGGTGAACTCCATGGATTGT-3′; the underlined bases are Nde I restriction site) and PH0540-R (5′-GGTGGTGGTGCTCGAGAGTTCCGTGCTCCCAGCT-3′; the underlined bases are Xho I restriction site). DNA amplification was performed using the KOD One® PCR Master Mix Blue (Toyobo, Osaka, Japan). The DNA fragments were ligated into the expression vector pET28b (Novagen, San Diego, CA, USA). DNA sequencing was performed using Eurofins Genomics (Tokyo, Japan). Expression and purification of the recombinant protein were performed as previously described (Yabuta et al.2015).
SDS-PAGE
SDS-PAGE was performed on 5%-20% (w/v) linear gradient polyacrylamide slab gels (ATTO Corporation, Tokyo, Japan). Proteins in the gel were stained with Coomassie Brilliant Blue.
Enzyme assay
SAHH activity was measured using a high-performance liquid chromatography (HPLC) method described by Lozada-Ramírez et al. (2013) with some modifications. Briefly, the assay mixture (125 µL) for SAHH activity contained 50 m m potassium phosphate buffer (pH 6.5), 2 m m Ado, 4 m m Hcy, and 2.5 µg of purified enzyme. The reaction was initiated by addition of the enzyme. Incubation was performed in microtubes at 60 °C for 5 min. The tubes were immediately cooled on ice, and the enzyme reaction was stopped by adding 12.5 µL of 3 N HClO4. The reaction mixture was then neutralized by the addition of 112.5 µL of 0.5 M phosphate buffer (pH 6.5) and subsequently filtered through a 0.45-µm membrane filter (DISMIC Syringe Filter, 03CP045AN, Advantec, Tokyo, Japan). A Shimadzu (Kyoto, Japan) HPLC apparatus (LC-10Ai pumps, SPD-10Avvp UV-visible detector, SCL-10A VP system controller, DGU-20A3R degassing unit, and CTO-10Avp column oven) were used to detect SAH formed from Ado and Hcy by SAHH. Aliquots (20 µL) of filtrate were applied to a reversed-phase HPLC column (Cosmosil 5C18-AR-II, Φ3.0 × 150 mm), which was equilibrated at 40 °C with 0.2 M potassium phosphate and methanol (98:2) at a flow rate of 1.0 mL min−1. SAH was isocratically eluted under the same conditions and monitored by measuring the absorbance at 260 nm. Under the experimental conditions, the retention times of SAH and Ado were 5.1 and 8.0 min, respectively.
LC-MS/MS analysis
Liquid chromatography-mass spectrometry/Mass spectrometry analysis was performed as previously described (Nishimura et al.2021) with some modifications. A Waters (Waters, Milford, MA, USA) LC-MS/MS apparatus (Quattro Micro API mass spectrometer and Acquity UPLC system) was used for the detection of Ado and SAH. The LC conditions were as follows: column: Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 µm (Waters); column temperature, 40 °C; flow rate, 0.2 mL min−1; solvents, 0.1% formic acid aq. (A), and 0.1% formic acid in acetonitrile (B); gradient, 1%-20% B/(A + B) within 5 min.
Thermostability
The purified SAHH (0.7 mg mL−1) was incubated in 50 m m potassium phosphate buffer (pH 6.5) at 55 ºC-95 °C in the presence or absence of 0.5 m m NAD+. Aliquots were removed after 10 min of incubation, and SAHH activity was determined.
Surface hydrophobicity analysis
Surface hydrophobicity (H0) was measured as described by Ge et al. (2017), with some modifications. The H0 of purified P. horikoshii SAHH was determined using 8-anilino-1-naphthalenesulfonate (ANS) ammonium salt (Tokyo Chemical Industry Co., Ltd.). The enzyme was diluted (0.02, 0.04, 0.06, 0.08, and 0.1 mg mL−1) in 50 mM potassium phosphate buffer (pH 6.5) with/without 0.5 m m NAD+ and the dilutions were incubated at 90 °C for 10 min. Then, 0.5 µL of 8 m m ANS was added to 100 µL of the enzymes. Fluorescence intensity at 495 nm was measured using a microtiter plate reader (Infinite® 200 pro, TECAN, Männedorf, Switzerland) with excitation at 370 nm. Surface hydrophobicity was calculated as previously described by Hou and Chang (2004).
Statistical analyses
All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS Statistics software (version 28; IBM, Armonk, NY, USA).
Results and discussion
Phylogenetic analysis of P. horikoshii SAHH
The P. horikoshii genome contains PH0540 gene, annotated as SAHH, which consists of 425 amino acid residues with a predicted molecular mass of 47.7 kDa. The deduced amino acid residues of PH0540 had a high identity with SAHH from Pyrococcus furiosus (95%) and Pyrococcus abyssi (93%), as observed by a comparison analysis using the BLAST program of the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov).
SAHH is widely distributed in various organisms and is responsible for maintaining the appropriate ratio of SAM to SAH in vivo. Phylogenetic analysis was performed to characterize P. horikoshii SAHH. Amino acid sequences of SAHH from various organisms were obtained from the Kyoto Encyclopedia of Genes and Genomes databases (https://www.genome.jp/kegg/kegg_ja.html) to construct a phylogenetic tree. Phylogenetic analysis showed that P. horikoshii SAHH was closely related to SAHH from thermophilic organisms (Figure 2). In particular, SAHHs of Saccharolobus solfataricus (formerly Sulfolobus solfataricus), Thermotoga maritima, and P. furiosus have been reported to be thermostable enzymes (Porcelli et al.2000; Porcelli et al.2005; Lozada-Ramírez et al.2013; Qian et al.2014). Thermosynechococcus elongatus inhabits hot springs and has an optimal growth temperature of ∼55 °C (Nakamura et al.2002). Although it is unclear, which sequences contribute to the thermostable surname, our findings suggest that there is a sequence specific to thermostable SAHH.
Phylogenetic analysis of SAHHs from various organisms. The NCBI protein IDs for enzymes used in the phylogenetic analysis are as follows: Arabidopsis thaliana, NP_193130; Caenorhabditis elegans, NP_491955; Corynebacterium efficiens, BAC17577; Danio rerio, NP_954688; Drosophila melanogaster, NP_001285268; Homo sapiens, NP_006612; Mus musculus, NP_057870; Oryza sativa, XP_015616900; Plasmodium falciparum, XP_001351767; Pseudomonas aeruginosa, NP_249123; Pyrococcus furiosus, AAL80467, Pyrococcus horikoshii, BAA29629; Rattus norvegicus, NP_058897; Rhodococcus jostii, ABG98099; Saccharolobus solfataricus, AAK41050, Saccharomyces cerevisiae, NP_010961; Schizosaccharomyces pombe, NP_595580; Thermosynechococcus vestitus, BAC09942; and Thermotoga maritima, AAD35265
Expression of the recombinant P. horikoshii SAHH in E. coli
The coding region of PH0540 was cloned into the pET28b vector and recombinant proteins with resulting N- and C-terminal hexahistidine tags were expressed in E. coli. P. horikoshii SAHH was expressed in the soluble fraction (data not shown) and purified to homogeneity using cobalt affinity chromatography. SDS-PAGE analysis of the purified enzyme revealed the presence of a single detectable protein band with a molecular weight of 50.9 kDa, which was calculated from the deduced amino acid sequence of PH0540 (Figure S1). The purified recombinant protein catalyzed the conversion of Hcy and Ado to SAH, as confirmed by LC-MS/MS analysis (data not shown). The specific activity of the enzyme was 122.9 ± 2.0 nmol min−1 mg−1 protein at 60 °C. SAHH is a homotetramer containing one tightly bound NAD(H) per subunit and catalyzes reactions without the need for additional cofactors (Palmer and Abeles 1979). P. horikoshii SAHH showed activity without the addition of NAD+, suggesting that it has similar properties.
Optimum temperature and thermostability of P. horikoshii SAHH
The optimum temperature for P. horikoshii SAHH was determined by carrying out enzymatic assays at varying temperatures ranging from 35 °C to 95 °C. We confirmed that Ado, Hcy, and SHA do not degrade after heating at 95 °C for 5 min. Surprisingly, the optimum temperature for the enzyme was 60 °C, which was considerably lower than the optimum temperature for P. horikoshii growth (González et al.1998). Remarkably, the optimal temperature for P. horikoshii SAHH was found to be 60 °C, which was lower than that for a similar ortholog from P. furiosus (Porcelli et al.2005).
The thermostability of P. horikoshii SAHH was also investigated. Heat treatment at just 55 °C markedly reduced SAHH activity (approximately 30.2%, Figure 3b) compared to its activity without heat treatment. The thermostability of P. horikoshii SAHH was therefore lower than that observed for P. furiosus and T. maritima SAHHs (Porcelli et al.2005; Lozada-Ramírez et al.2013).
Optimum temperature and thermostability of P. horikoshii SAHH. Optimum temperature (a) and thermostability (b) of P. horikoshii SAHH. Each value represents the mean of three independent measurements.
Booth et al. (2018) have reported that His-tag altered the thermostability of proteins and the effect was 0.2 ± 2.3 °C, which may also need to be considered. However, P. horikoshii SAHH may not be affected as strongly.
Activation of P. horikoshii SAHH by NAD+
The catalytic mechanism of SAHH involves a reciprocal oxidation-reduction cycle of substrate and enzyme-bound NAD(H) (Palmer and Abeles 1979). As described above, SAHH exhibits catalytic activity without requiring additional cofactors because NAD(H) is tightly bound to it (Palmer and Abeles 1979). However, Dictyostelium discoideum SAHH is activated by preincubation with NAD+, which has a marked effect on subunit–subunit interactions (Hohman et al.1984). In other words, NAD+ strengthens the bonds between subunits. It has been reported that the addition of NAD+ to the reaction mixture increased the activity of T. maritima SAHH and changed the optimal temperature (from 85 °C to 100 °C) and pH (from 8.0 to 11.2) of T. maritima SAHH (Qian et al.2014). Selwood and Jaffe (2012) reviewed several proteins, including D. discoideum SAHH, whose enzyme activity was altered by a change in quaternary structure (e.g. dimer to tetramer). Furthermore, Lupinus luteus and rat SAHHs with multiple oligomeric states have been reported (Guranowski and Pawelkiewicz 1977; Kajander and Raina 1981). Although the detailed mechanism of enzyme action remains unclear, these results suggest that addition of external NAD+ stabilizes the structure of SAHH and enhances enzyme activity. Other than these, there have been no reports on the thermal stability of SAHH with NAD+ addition. Therefore, we investigated the effect of NAD+ on the activity of P. horikoshii SAHH. The activity of P. horikoshii SAHH was dramatically enhanced by the addition of NAD+ (Figure 4). The specific activity at 60 °C in the presence of 5 m m NAD+ was 212.2 ± 1.4 nmol min−1 mg−1 protein, which was 1.66-fold higher than in the absence of NAD+. The molar ratio of NAD+ to enzyme was approximately 25000:1 when 10 m m NAD+ was added. Interestingly, in the presence of 0.5 m m NADH, the specific activity decreased to 54.8% (67.3 ± 1.5 nmol min−1 mg−1 protein) at 60 °C compared to that in the absence of NAD+.
Effect of NAD+ at different concentrations on the activity of P. horikoshiiSAHH. SAHH activity was measured at 60 °C. Each value represents the mean of three independent measurements.
Although the intracellular NAD+ concentration of P. horikoshii is still unknown, it has been reported that the intracellular NAD+ level of E. coli is 0.64 ± 0.04 m m (Zhou et al.2011). When the optimum temperature of P. horikoshii SAHH was examined in the presence of 0.5 m m NAD+, the optimum temperature for the enzyme was shifted to 65 °C (Figure 5a). Furthermore, SAHH activity was maintained above 65 °C and the thermostability of P. horikoshii SAHH was improved by the addition of NAD+ (Figure 5b). Addition of NAD+ at 0.5 m m after heat treatment of P. horikoshii SAHH at 95 °C did not restore the activity of the enzyme (6.7 nmol min−1 mg−1 protein). These findings suggest that SAHH functions in P. horikoshii cells, but with the help of cofactors such as NAD+.
Effect of NAD+ on optimum temperature and thermostability of P. horikoshii SAHH. Optimum temperature (a) and thermostability (b) of P. horikoshii SAHH in the presence of 0.5 m m NAD+. Each value represents the mean of three independent measurements.
The effect of NAD+ on the thermostability of the enzyme was also investigated using surface hydrophobicity analysis (Table 1). The surface hydrophobicity of P. horikoshii SAHH was measured after incubation at 4 °C (control) or 90 °C for 10 min. As shown in Table 1, addition of different concentrations of NAD+ affected the surface hydrophobicity of P. horikoshii SAHH. The surface hydrophobicity of P. horikoshii SAHH after 0.5 m m and 5 m m NAD+ addition was approximately 38.7% and 41.0% lower than that of the sample with no NAD+ addition, respectively. Our data indicate that the addition of NAD+ stabilizes the SAHH structure of P. horikoshii and improves its thermostability.
Effect of surface hydrophobicity values (H0) of P. horikoshii SAHH with and without NAD+
| Control | Heat treatment | |
|---|---|---|
| None | 16 090.0 ± 2866.6a,b | 34 430.0 ± 1814.2d |
| 0.5 m m NAD+ | 14 830.0 ± 567.0a | 21 113.3 ± 978.3c |
| 5 m m NAD+ | 13 850.0 ± 529.8a | 20 298.3 ± 1049.4b,c |
| Control | Heat treatment | |
|---|---|---|
| None | 16 090.0 ± 2866.6a,b | 34 430.0 ± 1814.2d |
| 0.5 m m NAD+ | 14 830.0 ± 567.0a | 21 113.3 ± 978.3c |
| 5 m m NAD+ | 13 850.0 ± 529.8a | 20 298.3 ± 1049.4b,c |
Values are the means ± SD (n = 3). Different letters indicate values that are significantly different (P < 0.05)
Effect of surface hydrophobicity values (H0) of P. horikoshii SAHH with and without NAD+
| Control | Heat treatment | |
|---|---|---|
| None | 16 090.0 ± 2866.6a,b | 34 430.0 ± 1814.2d |
| 0.5 m m NAD+ | 14 830.0 ± 567.0a | 21 113.3 ± 978.3c |
| 5 m m NAD+ | 13 850.0 ± 529.8a | 20 298.3 ± 1049.4b,c |
| Control | Heat treatment | |
|---|---|---|
| None | 16 090.0 ± 2866.6a,b | 34 430.0 ± 1814.2d |
| 0.5 m m NAD+ | 14 830.0 ± 567.0a | 21 113.3 ± 978.3c |
| 5 m m NAD+ | 13 850.0 ± 529.8a | 20 298.3 ± 1049.4b,c |
Values are the means ± SD (n = 3). Different letters indicate values that are significantly different (P < 0.05)
The variation of velocity as a function of substrate concentration showed typical Michaelis-Menten kinetics for Ado or Hcy. From the Lineweaver-Burk plot, the apparent KM values of the purified enzyme for Ado and Hcy were 1.71 m m and 84.5 µm, respectively (Table 2). The affinity of P. horikoshii SAHH for Ado was much lower than that of the enzymes from other sources, while the affinities for Hcy were similar (Porcelli et al.2000 and 2005; Lozada-Ramírez et al.2013). The reason for this discrepancy is not clear.
Kinetic parameters of thermostable SAHH from several organisms
| Km for Hcy (µm) | Km for Ado (µm) | |
|---|---|---|
| T. maritimaa | 135 | 49 |
| P. furiosusb | 140 | 4.8 |
| S. solfataricusc | 148 | 42 |
| P. horikoshiie | 84.5 | 1710 |
| P. horikoshii + 0.5 m m NAD+e | 5.6 | 520 |
Kinetic parameters of thermostable SAHH from several organisms
| Km for Hcy (µm) | Km for Ado (µm) | |
|---|---|---|
| T. maritimaa | 135 | 49 |
| P. furiosusb | 140 | 4.8 |
| S. solfataricusc | 148 | 42 |
| P. horikoshiie | 84.5 | 1710 |
| P. horikoshii + 0.5 m m NAD+e | 5.6 | 520 |
Interestingly, P. horikoshii SAHH KM values for these substrates decreased in the presence of 0.5 m m NAD+ (apparent KM values of the enzyme for Ado, and Hcy were 0.52 m m, and 5.6 µm, respectively). The crystal structures of human and rat SAHH have been previously determined (Turner et al.1998; Hu et al.1999). The subunit of SAHH consists of a catalytic domain, NAD+-binding domain, and small C-terminal domain (Turner et al.1998; Hu et al.1999). Hu et al. (1999) proposed that binding of SAH to the catalytic domain triggers a significant conformational change, bringing the ribose moiety of SAH in close proximity to the nicotinamide moiety of NAD. Thus, the proximity of NAD+ to the substrate has a significant effect on enzyme activity. Although the detailed mechanism is unclear, our findings support the hypothesis that the addition of NAD+ causes a conformational change in SAHH, affecting the substrate affinity and catalytic efficiency.
Six amino acid residues (E242, V223, I298, N345, K425, and Y298) of rat SAHH have been reported to interact with NAD+ (Hu et al.1999). Interestingly, the amino acid residues corresponding to V223, I298, K425, and Y298 of rat SAHH are not conserved in thermostable P. horikoshiim, P. furiosus, and T. maritima SAHHs (Figure S2), suggesting that these differences may be associated with the increase in thermostability and activation of P. horikoshii SAHH by NAD addition. Further detailed analysis is needed in the future.
Our findings suggest that to efficiently produce SAH from Ado and Hcy using P. horikoshii SAHH, NAD+ should be added and the reaction should be carried out at high temperatures. However, NAD is expensive. Qian et al. (2014) reported that NAD-dependent oxidoreductases, such as lactate dehydrogenase, can be used in combination to conserve the added NAD+.
Acknowledgments
We thank Prof. Haruhiko Sakuraba, Kagawa University, for the generous gift of P. horikoshii genomic DNA.
Data availability
The data underlying this article will be shared on a reasonable request to the corresponding author.
Author contribution
R.I. and Y.Y. performed the majority of the experiments. A.I. performed LC-MS/MS analysis. Y.Y. designed the experiments and interpreted the data. Y.Y., T.B., and F.W. wrote the manuscript. All authors commented on the manuscript and approved its final version.
Disclosure statement
No potential conflict of interest was reported by the authors.
