Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi

Catalysing the reversible interconversion of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamate in the presence of pyridoxal 5 phosphate, alanine aminotransferase (ALT) is an enzyme which operates at the cross roads of amino acid and carbohydrate metabolism. The enzyme has been reported from a wide range of organisms including animals, plants, fungi and microbes. The enzyme has a clinical applications in the diagnosis of many diseases. In the present study we have produced a recombinant of ALT from Pyrococcus abyssi in BL21 (DE3) strain of E. coli . The recombinant enzyme was purified by anion exchange chromatography, it displayed a 45kDa band on SDS-PAGE, with 58.1% final recovery, 15.3 fold purification and 139 U/mg specific activity. Maximum enzyme activity was found at pH 8 and above 90C, its K M and Vmax values were found 25µM L-alanine and 149 U/min/mg respectively. In silico studies have shown that the enzyme was found in a monomer structure. Molecular docking studies with potential molecules involved in the reaction catalysed have been conducted and binding energy values were calculated for each molecule including L-alanine, pyridoxal 5 phosphate, pyruvate, alpha ketoglutarate and L-glutamate. Present study provides the first report of ALT from Pyrococcus abyssi and suggests active site residues of enzyme from archaeal origin.


INTRODUCTION
A lanine aminotransferase (ALT), (EC 2.6.1.2) is the enzyme responsible to catalyse reversible conversion of L-alanine and alpha-ketoglutarate to L-glutamate and pyruvate in the presence of pyridoxal phosphate which acts as a prosthetic group. ALT catalysed reaction provides a link between the amino acid and carbohydrate metabolism by producing pyruvate (an intermediate of carbohydrate metabolism from L-alanine. There are two isoforms of ALT found in the human body, ALT1 or cytosolic form and ALT2 or mitochondrial form, both are coded by different genes. ALT2 is exclusively produced in the heart and skeletal muscles (Mendes-Mourao et al., 1975;Pan et al., 2003;Sato et al., 2004;Shan et al., 2009). ALT is ubiquitous in nature and studied from a wide range of organisms including Alternaria conidia, humans, algae such as Chlamydomonas reinhardtii, higher plants, and silk worm (Varley et al., 2002;Good et al., 2007;Liu et al., 2008;Good and Beatty, 2011;Inagaki et al., 2012;Lecler et al., 2012;De Linares et al., 2022). There are several clinical and industrial applications of ALT, as for example the human plasma levels of ALT have been used as a biomarker for liver damage or liver diseases (Kim et al., 2008). The enzyme has also been used for the diagnosis of some cancers and muscular dystrophy (Wagner et al., 2021;Muyas et al., 2022). The elevated levels of ALT in blood plasma has also been linked with diabetes, hyperlipidaemia, obesity and cardiovascular diseases (Yun et al., 2009;Bril et al., 2020;Kathak et al., 2022;Saleh et al., 2022). ALT also has applications in the urea producing and pharmaceutical industries (Schreiber et al., 2002;Zia et al., 2021). Increasing demands of ALT and other important enzymes require the recombinant production by heterologous gene expression in a suitable system, E. coli based microbial gene expression system provides an economic, timely and simple system for the production of recombinant enzymes (Backlund et al., 2008). The microbial system has a capacity to produce bulk quantities of target enzymes and proteins (Jonet et al., 2012;Jia and Jeon, 2016;Zhou et al., 2018). In the recent times, in silico studies have been used for the prediction of 3D O n l i n e F i r s t A r t i c l e protein structure, active site amino acids, binding affinities of substrates and potential inhibitors to the enzyme active site, and ultimately the selection of better enzyme, substrate and inhibitors (Kamble et al., 2019;Comaki et al., 2020). In the present study, we aimed to produce the recombinant ALT from Pyrococcus abyssi GE5 strain. The target species is an archaeon that grows at 90 • C under high pressure conditions (Erauso et al., 1993;Cohen et al., 2003). Recombinant of enzyme was produced in E. coli, purified by chromatographic techniques, evaluated by kinetics and in silico analysis.

Materials
All reagents, kits, chemicals, PCR kit, dNTPs, DNA ligation and restriction enzymes, plasmid vectors for cloning, DNA/protein electrophoresis, plasmid isolation, restriction and analysis kits, chromatography columns and reagents were purchased from Thermo Fisher and Sigma-Aldrich. Genomic DNA of archaeon was obtained from DSMZ Germany. All reagents and molecules were of molecular biology grade.

Gene synthesis, sub-cloning, expression, and purification
ALT gene from Pyrococcus abyssi GE5 consists of 1197bp open reading frame (ORF). In the ORF, there is a restriction site for NdeI (catatg), 147 bp at 5' end, downstream to the start codon (ATG) and a restriction site for NcoI (ccatgg) at 188 bp upstream to the stop codon. We found it difficult to subclone the complete open reading frame in pET 21a(+) vector. Hence, the gene was synthesized commercially from Macrogen (South Korea), it was made available in pUC57 vector (2710 bp) which has ampicillin resistance site (Fig. 1).
The gene harbouring plasmid was used for the transformation of DH-10 cells by using Thermo Scientific. InsTAclone PCR Cloning Kit. #K1214, the successful transformants were grown in LB broth containing 100µg/ml ampicillin to isolate plasmids. Plasmid were isolated using GeneJET Plasmid Miniprep Kit Catalogue number: K0502 (Thermo Fisher-Waltham, Massachusetts, United States). Procedure and instructions given by the manufacturer were followed. The plasmids were confirmed by restriction analysis with NdeI and BamHI to generate sticky ends of gene. Same pair of restriction enzymes was used for the restriction of pET21a (+) plasmid vector. ALT gene was ligated into pET21a (+) expression vector using T4 ligase (EL0011-Thermo Fisher -Waltham, Massachusetts, United States), under the conditions provided by the supplier. The recombinant plasmid pET21-alt + was confirmed by restriction analysis using NdeI and BamHI. The confirmed recombinant cells of BL21 (DE3) strain of E. coli were subjected to induction of gene expression. In short, the cells were grown in LB-broth supplemented with 100µg/ ml ampicillin, in the log phase of bacterial growth when the OD 600nm of bacterial culture was from 0.5 to 0.6, the expression of ALT gene was induced by the addition of 0.5mM IPTG (Isopropyl β-D-1-thiogalactopyranoside), final concentration in the medium. Induced E. coli culture was grown for 3.5 h at 37 • C and stored at 4 • C. The cells were harvested by centrifugation at 7000 rpm for 10 min at 4 • C. The cells were sonicated at a medium power for

O n l i n e F i r s t A r t i c l e
10 min by repeating 1 min exposure to incubation in the ice-box, alternatively. The cellular residue was removed by centrifugation at 12000 rpm/10 min at 4 • C, clear supernatant was retained and subjected to dialysis in 20mM phosphate buffer pH 7.5 adjusted at 4 • C (buffer A). The chromatography column (2.5 x 36 cm) was prepared by adding 30 mL of pre-soaked DEAE-Sephadex and equilibrated with buffer A. Dialyzed sample was clarified by centrifugation at 15000 rpm/10min at 4 • C and clear supernatant was loaded onto the column at a flow rate of 2.5 mL/min, unbound proteins were washed by using buffer A, enzyme was eluted with an increasing gradient (0 to 0.5M) of sodium chloride (NaCl), fractions of 2 mL were collected, and stored at 4 • C.

Characterization of recombinant ALT
All reagents and chemicals used for the enzyme assay were prepared in 20 mM phosphate buffer pH 7.5 (buffer A). The reagents included 1.2M L-alanine, 0.5M α-ketoglutarate, 5 to 7mg/mL of NADH disodium salt, lactate dehydrogenase 300 to 400 international units, and suitable dilution of purified enzyme. Before the measurement of enzyme activity, 100mg PLP was mixed well to 1mL of purified enzyme dilution. A UV/Vis spectrophotometer UV-1900i (Shimadzu Kyoto, Kyoto, Japan) was adjusted at 25 o C and 340 nm. A neat clean 2mL capacity glass cuvette was carefully added with 1 mL of L-alanine solution, 150 µL alpha-ketoglutarate, 100 µL NADH solution (freshly prepared), 100 µL of LDH and incubated for 5 min. At the end 10 µL of enzyme was added and mixed well by pipetting. Increase in the reading at 340 nm was noted for 3 min. Total protein content was determined byBradford et al. (1956) by using a standard curve prepared by using bovine serum albumin. The enzyme activity was calculated by the following formula: Enzyme activity was determined in the presence of buffer solutions adjusted at different pH values (4,5,6,7,8,9,10), and adjusting the temperature of reaction mixture at variable temperatures (from 30 o C to 90 o C); optimum pH and temperature for the enzyme activity were calculated. Enzyme stability was determined after 10 min of incubation of enzyme at different temperatures (60 o C, 70 o C, 80 o C, 90 o C, 100 o C) prior to enzyme assay keeping the other conditions constant. The enzyme activity was also calculated by regular increase in the substrate concentration under optimum pH and temperature conditions to determine the K M and V max values of enzyme by Lineweaver-Burk plot. Enzyme activity was also measured under optimum conditions in the presence of 100µM of copper (Cu), cadmium (Cd), iron (Fe), manganese (Mn), lead (Pb), chromium (Cr), magnesium (Mg), calcium (Ca), to determine the effect of these ions on the enzyme activity.

Protein modeling and validation
Protein structure of alanine aminotransferase from Pyrococcus abyssi was built using I-TASSER server (Yang et al., 2015), which develops 3D models from the structure templates identified by LOMETS from the PDB library. The templates of the highest significance in the threading alignments is selected by I-TASSER. Subsequently, the quality of the predicted model was examined by RAMPAGE, a protein structure validation server (Lovell et al., 2003;Wang et al., 2016), which reveals the results in terms of phi, psi and C-beta deviations by generating a Ramachandran plot for the protein built.

Structural alignment
After the structure assembly simulation, TM-align structural alignment program (Zhang and Skolnick, 2005;Madhusudhan et al., 2009), was employed to match the first I-TASSER model to all the structures in PDB library. Due to the structural similarity, these protein often have similar functions to the target. High TM-Score represents closest structural similarity.

Docking analysis
The 3D model was then prepared for docking analysis, by eliminating hetero atoms including water molecules and performing energy minimization by PyMol (Yuan et al., 2017;Faure et al., 2019), and ModRefiner (Xu and Zhang, 2011;Dhorajiwala et al., 2019), respectively. The PDB structures of selected ligands, i.e., alanine, alpha-ketoglutarate, pyruvate, glutamate and pyridoxal-5phosphate were downloaded from ChemSpider chemical database. All selected ligands were docked against the ALT-PA protein model using Hex docking server (Macindoe et al., 2010;Ritchie and Venkatraman, 2010). The ∆G (binding free energy) of each docked proteinligand complex was recorded to analyze the binding nature of the complexes.

Production, purification and properties of recombinant ALT
Under the T7 promoter system, a recombinant of ALT from P. abyssi was successfully generated in E. coli cells. It was purified up to 15.3 folds by anion exchange chromatography with more than 58% final recovery. The purified enzyme has shown a specific activity of 138 U/ mg of protein, it displayed a 45 kDa protein band on SDS-PAGE (Table I, Fig. 2).  Purified enzyme has shown activity at a wide range of temperatures and pH with an optimum activity at above 90•C and pH 8. More than 50% enzyme activity was retained when incubated at 100•C for 10 minutes indicating a high temperature stability. The measurement of activity in the presence of different metal ions has shown a significant increase in the enzyme activity in the presence of Mg 2+ ions. Purified ALT investigated in the present study has shown a K M value of 25 µM L-alanine and V max 149.25U/mg/min (Fig. 3).

Protein modeling and validation
I-TASSER generated the ALT-PA protein model has been generated (visualization by PyMOL Molecular Graphics System, Version 1.2r3pre.), by using 1XI9 as a homologous template, which determines the crystal structure of ALT from Pyrococcus furiosus. The quality assessment by Ramachandran plot reveals that the template-based structure of ALT from P. abyssi was quite stable, with >99% of residues in the favored and allowed regions (Fig. 4). The structural alignment results by TM-align shows 1xi9A to be ranked number 1 as the template crystal model (1XI9) contained identical residues in its protein sequence which are structurally 99% identical to that of query with lowest RMSD values when compared to other top PDB hits.

O n l i n e F i r s t A r t i c l e
Characterization of Gene Coding for Alanine Aminotransferase from P. abyssi 5 Docking and comparative analysis Docking results summarized in the Table II revealed that ALT-pyridoxal-5-phosphate complex (Fig. 5A) obtained the lowest free energy corresponding to highest binding affinity, followed by ALT-glutamate (Fig. 5C), ALT-alpha-ketoglutarate (Fig. 5E), ALT-L alanine (Fig.  5H) and ALT-pyruvate (Fig. 5I) docked complexes.  Ranking of proteins is based on TM-score of the structural alignment between the query structure and known structures in the PDB library. b Template Modeling Score (TM-score) measure the structural similarity between two structures TM-score >0.5 indicates a model of correct topology and a TM-score<0.17 means a random similarity range 0 to 1). c Heavy atoms Root-Mean-Square Deviation (RMSD) with respect to the experimental structure. d IDEN is the percentage sequence identity in the structurally aligned region (range 0 to 1. e Cov represents the coverage of global structural alignment and is equal to the number of structurally aligned residues divided by length of the query protein cluster. (range 0 to 1).

DISCUSSION
ALT is an enzyme of amino acid metabolism that also links the amino acid metabolism and carbohydrate metabolism. The enzyme is widely distributed in natural world such as in plant, animals, fungi and bacteria (Ward et al., 2000;Hosono et al., 2003;Duan et al., 2009;Good and Beatty, 2011). ALT has a great clinical importance, it has been extensively used as a biomarker for the diagnosis of liver diseases, liver injuries, it serves as a biosensor, diagnosis of dengue fever, detection of hepatitis B and C (Li et al., 2006;Senior, 2012;EASL, 2014;Gao et al., 2017;Wang et al., 2018;Ayaz and Furrukh, 2020). The present study was aimed at the recombinant production, purification, kinetics and in silico properties of enzyme from Pyrococcus abyssi which is a hyperthermophilic archaeon, unexplored for most of its enzyme, including ALT. The ALT coding gene has been investigated from a wide range of species including man to higher animals, and plants to bacteria. These genes coding for ALT in different species have shown different nucleotide sequences and properties of their protein products. All this information based on gene sequences and subsequent protein characteristics contribute to the pool of literature on ALT   126,127,128,131,132,360,361,362,102,103,207,233,234,235,236,241,242,243,244,245,246,247,112,113,114,115,168,260,261,172,173,174,175,188,204,205,206,207,208,209,217,218,219,220,101,102,103,106,127,205,207,236,237,245 Note: ∆G represents binding free energies of docked complex of respective protein and ligand. (Shrawat et al., 2008;Parthasarathy et al., 2019). In the present study, a gene coding for alanine aminotransferase from Pyrococcus abyssi (Fig. 1). has been cloned in E. coli and the properties of its corresponding recombinant protein have been investigated. The commercially synthesized gene was restricted out from pUC57 plasmid vector and introduced into pET 21a (+) expression vector, the later was used for the transformation of BL21 (DE3) strain of E. coli. The confirmed recombinant E. coli cells were grown to their early log phase with 0.5 to 0.6 optical density at 600nm and gene expression was induced in the presence of 0.5mM final concentration of IPTG. Similar IPTG concentrations have been successfully used for the production of recombinant heterologous proteins (Assadi-Porter et al., 2008;Dvorak et al., 2015). The expressed enzyme was purified by chromatography; it has shown a molecular weight of 44 kDa on SDS-PAGE (Fig. 2). The enzyme with molecular weight of 54 kDa, 45 kDa, 94 kDa, and 50 kDa has been reported from different animal and plant species (Orzechowski et al., 1999;Liepman and Olsen, 2001;Yang et al., 2002;Kendziorek et al., 2012). The enzyme being reported in the present report has shown optimum activity at pH 8, and above 90 o C. It was stable at a wide pH range and temperature up to 100 o C, Mg 2+ ions increased the enzyme activity, the values of K M and V max of enzyme was 25µM L-alanine and 149.25U/ mg, respectively (Fig. 3). Enzyme isolated from a similar species, Pyrococcus furiosus, has shown optimum activity at pH 6.5 to 7.8 and 95•C. However, the K M value for this enzyme was 2.8mM L-alanine was high as compared to the enzyme being reported from P. abyssi (Ward et al., 2000). K M values of 5.0 mM has also been reported from bacterial species (Oikawa, 2006). There is no information available about the effect of metal ions on the activity of ALT. 3D structure based on in silico studies has shown that the enzyme exists as a monomer (Fig. 4). A monomer structure of enzyme has been reported in literature (Duff et al., 2012). Molecular docking studies with potential substrate molecules have shown the active site residues and binding affinities of enzyme with these molecules (Fig.   5). The docking structures were verified by using different alignment tools (Table II). Docking studies have shown binding energy of ∆G -221.58 kJ/mol with pyridoxal-5phosphate, ∆G -170.84 kJ/mol with L-glutamate, ∆G -162.48 kJ/mol with alpha-ketoglutarate, ∆G -128.84 kJ/ mol with L-alanine, ∆G -112.58 kJ/mol with pyruvate (Table III). ALT active site residues of enzyme from E. coli reported in a study include Tyr15, Tyr37 and Tyr152, Arg18 and Tyr129 (Pena-Soler et al., 2014). However, there are only a few studies available about the active site analysis of bacterial and archaeal ALT, the information provided by the in silico analysis will provide a baseline for the future research in these lines.

CONCLUSIONS
The present study provides insights into the clinically important enzyme from an unexplored species. The enzyme investigated in the study was stable at high temperatures with maximum activity above 90 o C indicating its potential in the industries. In silico studies have demonstrated binding affinities of different potential substrate molecules to the enzyme active site. The information acquired from the computer based studies require further confirmation by crystal structure analysis in the presence of substrate and PLP.

O n l i n e F i r s t A r t i c l e
Characterization of Gene Coding for Alanine Aminotransferase from P. abyssi

Statement of conflict of interest
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Characterization of Gene Coding for Alanine Aminotransferase from P. abyssi O n l i n e