title: Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.
abstract: Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.
authors.affiliation: Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Mohd Adilin
authors.initials: MA
authors.lastname: Yaacob
authors.affiliation: Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Wan Atiqah Najiah Wan
authors.initials: WA
authors.lastname: Hasan
authors.affiliation: Enzyme and Microbial Technology Research Centre and Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Mohd Shukuri Mohamad
authors.initials: MS
authors.lastname: Ali
authors.affiliation: Enzyme and Microbial Technology Research Centre and Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Raja Noor Zaliha Raja Abdul
authors.initials: RN
authors.lastname: Rahman
authors.affiliation: Enzyme and Microbial Technology Research Centre and Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Salleh
authors.affiliation: Enzyme and Microbial Technology Research Centre and Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Mahiran
authors.initials: M
authors.lastname: Basri
authors.affiliation: Enzyme and Microbial Technology Research Centre and Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Thean Chor
authors.initials: TC
authors.lastname: Leow
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D010776:Photobacterium / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D001215:Asparaginase