Characterization of thermostable serine hydroxymethyltransferase for β-hydroxy amino acids synthesis

被引:0
作者
Ilma Fauziah Ma’ruf
Elvi Restiawaty
Syifa Fakhomah Syihab
Kohsuke Honda
机构
[1] Institut Teknologi Bandung,Doctoral Program of Chemistry, Faculty of Mathematics and Natural Sciences
[2] Institut Teknologi Bandung,Biochemistry Research Group, Faculty of Mathematics and Natural Sciences
[3] Institut Teknologi Bandung,Chemical Engineering Process Design and Development Research Group, Faculty of Industrial Technology
[4] Universitas Pendidikan Indonesia,Faculty of Sports and Health Education
[5] Osaka University,International Center for Biotechnology
[6] Universitas Pertamina,Department of Chemistry, Faculty of Science and Computer
来源
Amino Acids | 2023年 / 55卷
关键词
Serine hydroxylmethyltransferase; Cloning; Characterization; Synthesis of β-hydroxy amino acids;
D O I
暂无
中图分类号
学科分类号
摘要
β-hydroxy amino acids, such as serine, threonine, and phenylserine, are important compounds for medical purposes. To date, there has been only limited exploration of thermostable serine hydroxylmethyltransferase (SHMT) for the synthesis of these amino acids, despite the great potential that thermostable enzymes may offer for commercial use due to their high stability and catalytic efficiencies. ITBSHMT_1 (ITB serine hydroxylmethyltransferase clone number 1) from thermophilic and methanol-tolerant bacteria Pseudoxanthomonas taiwanensis AL17 was successfully cloned. Biocomputational analysis revealed that ITBSHMT_1 contains Pyridoxal-3′-phosphate and tetrahydrofolatebinding residues. Structural comparisons show that ITBSHMT_1 has 5 additional residues VSRQG on loop near PLP-binding site as novel structural feature which distinguish this enzyme with other characterized SHMTs. In silico mutation revealed that the fragment might have very essential role in maintaining of PLP binding on structure of ITBSHMT_1. Recombinant protein was produced in Escherichia coli Rosetta 2(DE3) in soluble form and purified using NiNTA affinity chromatography. The purified protein demonstrated the best activity at 80 °C and pH 7.5 based on the retro aldol cleavage of phenylserine. Activity decreased significantly in the presence of 3 mM transition metal ions but increased in the presence of 30 mM β-mercaptoethanol. ITBSHMT_1 demonstrated Vmax, Km, Kcat, and Kcat/Km at 242 U/mg, 23.26 mM, 186/s, and 8/(mM.s), respectively. The aldol condensation reaction showed the enzyme’s best activity at 80 °C for serine, threonine, or phenylserine, with serine synthesis showing the highest specific activity. Biocomputational analysis revealed that high intramolecular interaction within the 3D structure of ITBSHMT_1 might be correlated with the enzyme’s high thermal stability. The above data suggest that ITBSHMT_1 is a potential and novel enzyme for the production of various β-hydroxy amino acids.
引用
收藏
页码:75 / 88
页数:13
相关论文
共 455 条
[1]  
Angelaccio S(2013)Extremophilic SHMTs: from structure to biotechnology BioMed Res Int 278 41789-41797
[2]  
Angelaccio S(2003)Catalytic and thermodynamic properties of tetrahydromethanopterin-dependent serine hydroxymethyltransferase from methanococcus jannaschii J Biol Chem 13 1314-1326
[3]  
Chiaraluce R(2012)Serine Hydroxymethyltransferase from the cold adapted microorganism psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity Int J Mol Sci 110 171-177
[4]  
Consalvi V(2014)Structural stability of cold-adapted serine hydroxymethyltransferase, a tool for β-hydroxy-α-amino acid biosynthesis J Mol Catal B Enzym 49 359-378
[5]  
Buchenau B(2012)Purification and characterization of a thermostable lipase from geobacillus thermodenitrificans IBRL-nra Enzyme Res 278 40793-40805
[6]  
Giangiacomo L(1985)Regulation of cytoplasmic pH in bacteria Microbiol Rev 279 504-514
[7]  
Bossa F(2003)Unusual structural, functional, and stability properties of serin hidroksimetiltransferase from J Biol Chem 268 6508-6525
[8]  
Constabile R(2011)Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase FEBS J 61 3891-3898
[9]  
Angelaccio S(2001)l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase Eur J Biochem 100 2579-2590
[10]  
Florio R(2021)AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings J Chem Inf Model 278 2645-2653