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Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis.
Int J Biol Macromol. 2018 Oct 01; 117:1216-1223.IJ

Abstract

A novel glyA gene was screened from a marine bacterium, Idiomarina loihiensis encoding a thermo-stable serine hydroxymethyl transferase (SHMT; 418 AA; 45.4 kDa). The activities of wild type (WT) and mutants were analyzed against d-phenylserine using pyrodoxal-5-phosphate (PLP) as cofactor under optimized conditions. Based on homology modelling and molecular docking, several residues were found that may be able to improve the activity of WT-SHMT. Site directed mutagenesis was conducted. The activity and thermostability of the wild type SHMT was improved by two variants H61G and G132P, which showed a noteworthy change in the thermo-stability and activity as compared to WT. To investigate the mechanism of activity of mutants, we combined two residues into one mutant DUAL. WT showed the optimum activity at 50 °C, whereas H61G, G132P and DUAL had the temperature optima of 55, 60 and 60 °C, respectively. These mutants G132P, H61G and DUAL were quite stable at 45 and 55 °C as compared to WT. Dual mutant was relatively more stable at all tested pH(s) while WT loses its activity in alkaline pH(s). Kinetics studies indicated the 1.52, 2.42 and 4.54 folds increase in the kcat value of H61G, G132P and Dual mutants as compared to WT respectively. The molecular docking indicated that hydrophobic interactions are more prominent than hydrogen-bonding and had more influence on ligand binding and active site cavity. The molecular dynamics showed the changed RMSD values for ligand and formation of new hydrogen bonds, hydrophobic interaction which considerably increased the activity and thermo-stability of the mutant proteins as compared to WT. Thus, increased stabilities at higher temperatures and activities can be attributed to new hydrogen bonding, altered active site geometry and increased ligand interactions.

Authors+Show Affiliations

College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China; Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 172 234, Himachal Pradesh, India.College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China.College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China.Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 172 234, Himachal Pradesh, India.College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430 070, China. Electronic address: lzd@mail.hzau.edu.cn.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29727646

Citation

Kumar, Ashok, et al. "Improved Catalytic Properties of a Serine Hydroxymethyl Transferase From Idiomarina Loihiensis By Site Directed Mutagenesis." International Journal of Biological Macromolecules, vol. 117, 2018, pp. 1216-1223.
Kumar A, Wu G, Wu Z, et al. Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis. Int J Biol Macromol. 2018;117:1216-1223.
Kumar, A., Wu, G., Wu, Z., Kumar, N., & Liu, Z. (2018). Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis. International Journal of Biological Macromolecules, 117, 1216-1223. https://doi.org/10.1016/j.ijbiomac.2018.05.003
Kumar A, et al. Improved Catalytic Properties of a Serine Hydroxymethyl Transferase From Idiomarina Loihiensis By Site Directed Mutagenesis. Int J Biol Macromol. 2018 Oct 1;117:1216-1223. PubMed PMID: 29727646.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis by site directed mutagenesis. AU - Kumar,Ashok, AU - Wu,Gaobing, AU - Wu,Zuo, AU - Kumar,Narendra, AU - Liu,Ziduo, Y1 - 2018/05/01/ PY - 2017/11/13/received PY - 2018/04/05/revised PY - 2018/05/01/accepted PY - 2018/5/5/pubmed PY - 2018/11/15/medline PY - 2018/5/5/entrez KW - Ligand binding KW - Molecular docking KW - Mutant KW - Site directed mutagenesis KW - Thermostability SP - 1216 EP - 1223 JF - International journal of biological macromolecules JO - Int J Biol Macromol VL - 117 N2 - A novel glyA gene was screened from a marine bacterium, Idiomarina loihiensis encoding a thermo-stable serine hydroxymethyl transferase (SHMT; 418 AA; 45.4 kDa). The activities of wild type (WT) and mutants were analyzed against d-phenylserine using pyrodoxal-5-phosphate (PLP) as cofactor under optimized conditions. Based on homology modelling and molecular docking, several residues were found that may be able to improve the activity of WT-SHMT. Site directed mutagenesis was conducted. The activity and thermostability of the wild type SHMT was improved by two variants H61G and G132P, which showed a noteworthy change in the thermo-stability and activity as compared to WT. To investigate the mechanism of activity of mutants, we combined two residues into one mutant DUAL. WT showed the optimum activity at 50 °C, whereas H61G, G132P and DUAL had the temperature optima of 55, 60 and 60 °C, respectively. These mutants G132P, H61G and DUAL were quite stable at 45 and 55 °C as compared to WT. Dual mutant was relatively more stable at all tested pH(s) while WT loses its activity in alkaline pH(s). Kinetics studies indicated the 1.52, 2.42 and 4.54 folds increase in the kcat value of H61G, G132P and Dual mutants as compared to WT respectively. The molecular docking indicated that hydrophobic interactions are more prominent than hydrogen-bonding and had more influence on ligand binding and active site cavity. The molecular dynamics showed the changed RMSD values for ligand and formation of new hydrogen bonds, hydrophobic interaction which considerably increased the activity and thermo-stability of the mutant proteins as compared to WT. Thus, increased stabilities at higher temperatures and activities can be attributed to new hydrogen bonding, altered active site geometry and increased ligand interactions. SN - 1879-0003 UR - https://www.unboundmedicine.com/medline/citation/29727646/Improved_catalytic_properties_of_a_serine_hydroxymethyl_transferase_from_Idiomarina_loihiensis_by_site_directed_mutagenesis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0141-8130(17)34455-0 DB - PRIME DP - Unbound Medicine ER -