Molecular architecture of pterin deaminase from Saccharomyces cerevisiae NCIM 3458

Introduction

Pteridine research has been renowned as eminent for scores of natal process [1]. It also acts as essential cofactors in the process of cell metabolism and has turned into a focal point of cancer screening research because of reported varying pteridine levels in cancers [2]. In the pathway of pteridines, one of the most momentous uncharted enzymes was pterin deaminase. Most of the studies have focused only on the pteridines and its derivatives, whereas the studies on pterin deaminase are countable. Pterin deaminase is an enzyme belonging to the family of hydrolases, acting on carbon-nitrogen bonds other than peptide bonds. The reaction took place as follows [3].

2−amino−4−hydroxypteridine+H2O⇌2,4−dihydroxypteridine+NH3

The pterin deaminases were disseminated as intracellular, membrane-bound or extracellular enzyme in a spacious variety of prokaryotic and eukaryotic sources [4]. The first report on the intracellular pterin deaminase was isolated from the bacterium Alcaligenes metalcaligenes by Levenberg and Hayaishi in 1959 [5]. However, extracellular pterin deaminase was reported in eukaryotic organisms such as Aspergillus sp., Mucor sp., Rhizopus sp. and Penicillium sp. [6], [7], [8]. Rembold and Simmersbach [9] revealed that the pterin deaminase present in rat liver showed the highest substrate specificity. Besides this report in mammalian system, there are no other research findings in mammalian pterin deaminase. The 3D structure of the pterin deaminase enzyme is not available in the database plow at present. This uncharacteristic pterin deaminase is lighting an interest in developmental and neuronal biologist. The structure and function of the pterin deaminase enzyme might prove to pave the way for resolving unsolved quests in the biological systems. Saccharomyces cerevisiae, being utilized as a model system by pioneers of powerful functional genomics, systems and synthetic biology for recombination and fermentation technology [10], was selected as the test organism in the present study. This study was divulged to produce and purify enzyme pterin deaminase from S. cerevisiae 3458. In addition, the study involved the structure prediction of pterin deaminase through bioinformatic tools.

Materials and methods

Results

Purification of pterin deaminase

The ethanol precipitated enzyme, after suspension with potassium phosphate buffer, was estimated to have 44.60 U/mL of pterin deaminase activity, and protein content was measured to be 0.96 mg/mL. The specific activity was increased from 35.42 to 46.45 U/mg, and the purification fold was increased to 1.311 (Table 1).

Table 1:

Purification step of pterin deaminase.

Purification steps	Volume, mL	Enzyme activity, U/mL	Total protein, mg/mL	Specific activity, U/mg	Purification (fold)
Crude	40	38.97	1.1	35.42	1
Ethanol precipitation	10	44.60	0.96	46.45	1.311
Trap Q FF (ion-exchange column)	1	82.8	0.26	310.11	8.76

The ion exchange column purification process increased the purity of the enzyme, which was revealed by the increase in enzyme activity to 82.8 U/mL with the specific activity of 310.11 U/mg. The purification fold was increased to 8.76. Although the chromatogram revealed five active peaks, the fractions from 12 to 15 under the peak alone exhibited enzyme activity corresponding to 20% NaCl gradient solution (Table 1; Figure 1).

Figure 1:

Ion exchange chromatogram of purified pterin deaminase.

The protein profile of the crude enzyme showed more than four bands and appeared very thin due to very low concentration of loaded protein. The precipitated protein sample showed similar band pattern as crude but denser conforming the increased concentration of protein. Single band was visualized upon PAGE analysis of the active fractions (pooled) of ion exchange chromatography, and apparent molecular weight of the enzyme was approximately 60 kDa (Figure 2).

Figure 2:

SDS-PAGE (silver staining): lane 1, marker; lane 2, crude (20 μL); lane 3, ethanol precipitation (30 μL); lane 4, purified fraction (35 μL); lane 5, purified fraction (40 μL); lane 6, purified fraction (45 μL); lane 7, purified fraction (50 μL).

Confirmation of pterin deaminase

For further confirmation, the enzyme sample was run on 12% Native-PAGE at 50 V for 3 h. Upon incubation with the substrate (folic acid), the gel revealed fluorescence under UV light and confirmed the presence of pterin deaminase in the purified sample. The fluorescence observed was attributed to the presence of product lumazine, which has blue fluorescence. The band was clearly visible in precipitated protein where the protein concentration was higher (Figure 3).

Figure 3:

Activity staining of purified pterin deaminase: lane 1, FPLC purified fraction; lane 2, FPLC purified fraction; lane 3, ethanol precipitate.

Optimization of purified pterin deaminase

The optimum temperature was found to be 30 °C, which corresponded to the maximum enzyme activity of 51 U/mL (Figure 4), whereas alkaline pH 8 was optimized condition for pterin deaminase activity (Figure 5). Double reciprocal plot showed the K_m as 10 mM of folic acid and V_max of 317.30 U/mg (Figure 6). The overall characteristic of pterin deaminase was summarized in Table 2.

Figure 4:

Effect of temperature on enzyme activity.

Figure 5:

Effect of pH on enzyme activity.

Figure 6:

Double reciprocal plot.

Table 2:

Characteristics of purified pterin deaminase from Saccharomyces cervisiae.

Sample no.	Parameters	Units
1	Optimum pH	8
2	Optimum temperature °C	30 °C
3	Molecular weight	Approximately 60 kDa
4	Km, μM	10 mM
5	Vmax, IU/mg	317.30

Effect of inhibitors

To predict the nature of the enzyme, inhibitor studies were carried out with purified enzyme. The enzyme was inhibited by EDTA and 1,10-phenanthroline at 10 mM concentration (Figure 7), and inhibitors such as DTT and PMSF also partially inhibited the enzyme activity at 10 mM concentration. EDTA disodium salt inhibited the enzyme activity, which indicated that the enzyme to be a metallo enzyme.

Figure 7:

Effect of inhibitors on pterin deaminase activity.

Homology modeling of protein sequence

Pterin deaminase sequence from Agrobacterium radiobacter K84 was retrieved from NCBI GenBank (GI: WP_012652141.1). Domain region of retrieved sequence was aligned with Saccharomyces genome sequence and found 47% similarity matches with pterin deaminase (Figure 8). Aligned sequence was then subjected to translate for six frame translations, which have high consensus similarity. 1AF2, 1ALN, 1CTT, 1CTU, 1JTK, 1MQ0, 1A4L, 1A4M, 1ADD, 1FKW, 1FKX and 1KRM as PDB ID were chosen as a template to predict structure of pterin deaminase (Table 3). All the template selection was kept on the basis of functionally similar as deaminase activity with metals such as Mg, zinc and copper. Knowledge-based homology modeling runs as high complexity and low sequence identity alignment between query and subject from irrespective of organism specification and enzyme classification. Further templates were utilized to predict the structure using prime module from Schrodinger suite. The modeled structure was then hypothesized to undergo energy minimization to optimize geometrical conformations. Modeled protein structure was plotted against Ramachandran Rotomer library conformations and screened, allowed and disallowed region of amino acids stability (Figure 9).

Figure 8:

Alignment matrix of target sequence (WP_012652141) and query sequence (TRS85_851686).

Table 3:

List of DNA and protein sequences and template structures retrieved from databases.

Sample no.	Sample	ID	Organisms	Database	Details
1	DNA sequence	WP_012652141.1	Agrobacterium radiobacter K84	NCBI	Gene imparted with pterin deaminase activity as per earlier report (Fan et al. [18])
2	DNA sequence	TRS85 Trs85p	Saccharomyces cerevisiae	NCBI gene	Resultant gene sequence when gene of Agrobacterium radiobacter K84 was blasted against Saccharomyces cerevisiae
3	DNA	NC_001143.9	Saccharomyces cerevisiae	NCBI genome	Whole genome
4	Template protein structure	1A4L_A (adenosine deaminase)	Mus musculus	PDB	Ada structure complexed with deoxycoformycin at pH 7
		1A4M_A (adenosine deaminase)	Mus musculus	PDB	Ada structure complexed with purine riboside at pH 7
		1ADD_A (adenosine deaminase)	Mus musculus	PDB	A pre-transition state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deaza-adenosine and zinc-activated water
		1AF2_A (cytidine deaminase)	Escherichia coli	PDB	Crystal structure of cytidine deaminase complexed with uridine
		1ALN_A (cytidine deaminase)	Escherichia coli	PDB	Crystal structure of cytidine deaminase complexed with 3-deazacytidine
		1CTT_A (cytidine deaminase)	Escherichia coli	PDB	Transition-state selectivity for a single oh group during catalysis by cytidine deaminase
		1CTU_A (cytidine deaminase)	Escherichia coli	PDB	Transition-state selectivity for a single oh group during catalysis by cytidine deaminase
		1FKW_A (adenosine deaminase)	Mus musculus	PDB	Murine adenosine deaminase (d295e)
		1FKX_A (adenosine deaminase)	Mus musculus	PDB	Murine adenosine deaminase (d296a)
		1JTK_A (cytidine deaminase)	Bacillus subtilis	PDB	Crystal structure of cytidine deaminase from Bacillus subtilis in complex with the inhibitor tetrahydrodeoxyuridine
		1KRM_A (cytidine deaminase)	Bos taurus	PDB	Crystal structure of bovine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine riboside
		1MQ0_A (cytidine deaminase)	Homo sapiens	PDB	Crystal structure of human cytidine deaminase
5	Ligand	CID 6037	-	PubChem	Folic acid

Figure 9:

Plotting modeled amino acids conformations for stereo chemical quality and protein moiety check with Z-score analysis.

Interaction profile of modeled protein

Interaction profile of modeled pterin deaminase with folic acid showed that the ligand interacted at three sites of the protein having residual atom types ARG 370 (H), ARG 370 (H), ASN 337 (O) receptor via O (oxygen) and H (hydrogen) atom types forming oxygen and hydrogen bonds with the bond distance 2.214, 1.547 and 2.417 A, respectively (Figure 13). ARG and ASP have Schiff base and mediate meta portion amino group in the folic acid structure. Higher interaction of model protein versus folic acid obtained the glide score of −12.94 as higher interaction. Docking studies revealed that the protein sequence had folic acid binding capacity. Hence, using this as the base sequence of the gene responsible for pterin deaminase in S. cerevisiae 3458 further recombination and expression studies could be performed to confirm the function of the gene sequence. Analog conformations of folic acids interacted with metal binding proteins. Adenosine (Figure 10), guanine (Figure 11) and cysteine deaminase (Figure 12) proteins were selected to undergo docking procedure as to confirm functionality of deaminase. All obtained docking scores are lower than modeled pterin deaminase protein, i.e. −11.9, −4.1 and −8.3, respectively. Arginine is common amino acids that have interaction with all folic binding deaminase proteins.

Figure 10:

Interaction profile of folic acids and adenosine deaminase in the functional site as ribbon representation.

Figure 11:

Interaction profile of folic acids and cytosine deaminase in the functional site as ribbon representation.

Figure 12:

Interaction profile of folic acids and guanine deaminase in the functional site as ribbon representation.

Discussion

Saccharomyces cerevisiae is the most meticulously investigated eukaryotic microorganism and has been used in the production of food and alcoholic beverages, and these days this organism is also used in a number of different processes within the pharmaceutical industry [19]. Thus, it is now possible to introduce the production of anticancer enzyme pterin deaminase from this persuasive organism. Saccharomyces cerevisiae (NCIM 3458) has been so far explored for the production of aminohydrolase enzymes such as L-asparaginase and allantoinase, whereas this is the first attempt to pronounce the presence of pterin deaminase in the organism.

Folic acid, which is otherwise named as pteroyl glutamic acid, has pterin ring attached to glutamic acid by para amino benzoic acid. Hence, folic acid was used as substrate for the enzyme, which acts on amino group at the second position of the pterin ring. The report of Levenberg and Hayaishi [5] used pteroyl glutamic acid as the substrate in A. metalcaligenes, whereas Rembold and Simmersbach [9] reported isoxanthopterin, tetrahydropterin and pterin as substrates of pterin deaminase.

During the extraction process in this study, the higher protein precipitation was achieved at 1:9 ratios of protein-ethanol, and the purification fold was increased from 35.42 to 46.45 U/mg. Although the protein precipitation was reported to be complete and instantaneous with maximum recovery at all temperatures [20], only at lower temperature recovery of activated protein is a maximum that varies with pH [21]. Hence, the precipitation of the enzyme with 90% cold ethanol was substantiated.

Literally, there were no reports based on the purification of pterin deaminase enzymes in yeast, whereas Kusakabe et al. [7] reported the enzyme was purified from fungi Aspergillus Y8-5 (ATCC 20413) by DEAE cellulose column chromatography and obtained 84,975 units of pterin deaminase with the enzyme activity 825 U/mg of powder (1428 U/mg of protein). In this view, in the present study, DEAE cellulose column has been used from which the enzyme was eluted earlier at around 20% NaCl concentration with 10 mM phosphate buffer of pH 7. This indicated that the protein is negatively charged with less net charge, as binding with the matrix is not strong requiring only low salt concentration for its elution. In this study, the binding buffer of pH 7.0 has been used for the purification of this enzyme of alkaline nature as it was proved that 7.6 binding buffer resulted in higher resolution peaks for alkaline enzyme than those with higher pH binding buffer [22].

The molecular weight of the pterin deaminase was found to be 110 kDa in Bacillus megaterium [23]. In our study, the presence of pterin deaminase in S. cerevisiae was apparently the molecular weight was 60 kDa, which is similar to the previous report of Fan et al. [18] 47 kDa in Agrobacterium sp.

Confirmation of the presence of the enzyme was achieved by activity staining. The fluorescent property of the substrate and product was exploited for locating the enzyme in the native gel [5]. Upon incubation with the substrate and subsequent washing, the unbound substrate was removed leaving only the enzyme bound compounds. As both the substrate and product possess fluorescent capacity, the appearance of fluorescent band in native gel was attributed to the presence of enzyme illuminated by bound substrate or product.

The pterin deaminase characterized so far has been found to be active at alkaline pH [23], [24], [25], [26] owing to the release of ammonia during the reaction, and this has been confirmed in the present study also. Notably, this enzyme has been observed to be stable at higher temperature of 100 °C with around 10% of residual enzyme activity. The thermostability of the enzyme is imperative as the deamination process is an exothermic reaction, which requires the catalyzing enzyme to be thermostable [7]. Thus, the enzyme pterin deaminase was proven to be alkaline and thermostable enzyme. In earlier reports, pH 8 was measured as optimized condition for pterin deaminase activity, and it was capable of tolerating a pH range of 6.5–8.5 and also temperatures up to 90 °C [8], [22], [24], [25], [26], [27], [28].

Folic acid, an efficient substrate, was prepared for substrate kinetic determination. Elevation activity of folic acid and pterin deaminases was higher in S. cerevisiae (K_m 10 mM and V_max 317.30 IU/mg) than other prokaryotic organisms. Similarly, pterin deaminase catalyzing the hydrolytic deamination of pteridines was found in the bacterium, and B. megaterium showed the K_m value for 6-carboxypterin at 1.3 mM [23]. The K_m value of the enzyme from slime mold Dictyostelium discoideum strain Ax-2 was established as 0.002 mM folic acid for isoform I and for isoform II as 0.023 mM and 0.03 mM pterin. The pterin deaminase extracted from rat liver exhibited a K_m value of 30 μM for pterin [9]. The wide variation in K_m value may be attributed to the diversified sources (slime mold, bacteria and rat), which might be having varied concentration of substrate (pterin derivatives) metabolism.

Scientific reports suggest that the research groups were unable to identify efficient inhibitors for pterin deaminase. In spite of these, some of the inhibitors have been used and identified for the characterization of pterin deaminase in S. cerevisiae. The report of this study showed that the enzyme was deprived by EDTA and 1,10-phenanthroline at 10 mM concentration. Similarly, Levenberg and Hayaishi [5] found p-Cl-Hg benzoate and sodium or potassium fluoride caused severe inhibition for pterin deaminase activity when compared with other halide ions. In 1981, Wurster et al. [29] analyzed that the kinetic data for pterin deamination in the presence of 0.05 μM folic acid yielded typical competitive inhibition. Kim et al. [30] revealed KCN inhibited dihydropterin deaminase activity strongly (98.6%). Inhibition studies of the enzymes revealed some structural characters of the enzyme. The inhibition of enzyme activity by 1,10-phenanthroline demonstrates that the enzyme has metal component at 1,10-phenanthroline, forming strong complexes with zinc ions [31]. Further, the inhibition of the enzyme by EDTA corroborates the involvement of metal ion in the enzyme activity as EDTA chelates with heavy metals [32]. Interference of DTT in enzyme activity may be attributed to the presence of cysteine disulfide bonds in the enzyme as DTT reduces the disulfide bonds. Less inhibition of DTT might be interpreted as less disulfide bonds exposed to reducing reaction as DTT cannot reduce the disulfide bonds buried in the folds of the proteins [33], [34]. The reduced impact of PMSF on the enzyme activity portends the presence of less serine residues in the active site of enzyme activity as PMSF reacts with only hyperactive serine residues at the active site [35]. Thus, an inhibitory study prophesies the pterin deaminase to be a metallo enzyme with disulfide bridges of cystein and a few hyperactive serine residues.

The role of pterin deaminase in the body metabolism of S. cerevisiae is unknown, which may be revealed by silence gene mutation for which the gene sequence has to be determined. In this view, the present study was attempted to predict the gene sequence of the enzyme. Besides, this study added a further significant unique result of the protein structure of pterin deaminase in eukaryotic organisms S. cerevisiae. The reported gene sequence of pterin deaminase of A. radiobacter K84 was taken as the reference sequence that was used to pick out the pterin deaminase gene in S. cerevisiae genome. The protein structure of pterin deaminase was predicted and validated using Prime modules of Schrodinger suit 2016. The modeled structure of pterin deaminase and other folic binding proteins was interacted using glide modeling followed by extra precision method and rigid docking parameter. Folic acid is used as a substrate interacted with modeled pterin deaminase structure with Extra-Precision energy composition from Glide procedure. The higher interaction of model protein obtained the glide score −12.94. Docking score showed that it had good interaction with the enzyme, thus endorsing the functional property of pterin deaminase activity [18]. The marginal amount of score to consider is −12.94, and it hosts functional properties with folic complexity. Hence, the modeled pterin deaminase was proved for the ID: TRS85_851686 (Figure 13) of S. cerevisiae from proposed interaction profile, and the pterin deaminase structure can be taken into account for further biological activity. More recently, Fan et al. [18] determined a multidisciplinary approach that integrated homology modeling, molecular docking screens of a metabolite library and physical library screening by kinetic assays in Arad3529 from A. radiobacter K84 regarding pterin deaminase.

Figure 13:

Interaction profile of folic acids and modeled pterin deaminase in the functional site as ribbon representation.

Conclusion

Therapeutically, enzymology offers a means of treatment in tumors, although certainly insufficiently investigated to date. This study revealed novel data about the structural characterizes of pterin deaminase from S. cerevisiae. This study highlighted the possibility of extracting pterin deaminase enzyme from S. cerevisiae 3458 and also shed light on the biochemical properties of the enzyme activity. The confirmation of the predicted structure of pterin deaminase protein might lead to better understanding of the biological functions of this enzyme. Moreover, this study summarized herein appears to the first report of pterin deaminase from S. cerevisiae, and this may just open the windows for new drugs for cancer therapy.