title: Identification of active site residues of Fenugreek β-amylase: chemical modification and in silico approach.
abstract: The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India. Electronic address: garimasri99@gmail.com.
authors.forename: Garima
authors.initials: G
authors.lastname: Srivastava
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India. Electronic address: officerbiotech.1@gmail.com.
authors.forename: Vinay K
authors.initials: VK
authors.lastname: Singh
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India. Electronic address: kayasthabhu@gmail.com.
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D020134:Catalytic Domain; D062105:Molecular Docking Simulation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D029910:Trigonella / Q000201:enzymology / Q000235:genetics; D001614:beta-Amylase / Q000737:chemistry / Q000235:genetics
chemical_list: D010940:Plant Proteins; D001614:beta-Amylase
keywords: Active site; Chemical modification; Docking; Homology modelling; Trigonella foenum-graecum; β-Amylase