Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

Noor M. Shaik, Anurag Misra, Somesh Singh, Amol B. Fatangare, Suryanarayanarao Ramakumar, Shuban K. Rawal, Bashir M. Khan

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45 C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K m and V max were found to be 38.59 μM and 0.8237 μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037 μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

Original languageEnglish (US)
Pages (from-to)1351-1363
Number of pages13
JournalMolecular Biology Reports
Volume40
Issue number2
DOIs
StatePublished - Feb 2013
Externally publishedYes

Fingerprint

Glucosidases
Glucosides
Enzymes
Glycosides
Amygdalin
Hydrolases
Affinity Chromatography
Flavonoids
Catalytic Domain
Software
Binding Sites
Escherichia coli
Temperature
Genes
rhodiocyanoside A

Keywords

  • Glycosyl hydrolase family 1
  • Homology modeling
  • Leucaena leucocephala
  • Molecular docking

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul). / Shaik, Noor M.; Misra, Anurag; Singh, Somesh; Fatangare, Amol B.; Ramakumar, Suryanarayanarao; Rawal, Shuban K.; Khan, Bashir M.

In: Molecular Biology Reports, Vol. 40, No. 2, 02.2013, p. 1351-1363.

Research output: Contribution to journalArticle

Shaik, Noor M. ; Misra, Anurag ; Singh, Somesh ; Fatangare, Amol B. ; Ramakumar, Suryanarayanarao ; Rawal, Shuban K. ; Khan, Bashir M. / Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul). In: Molecular Biology Reports. 2013 ; Vol. 40, No. 2. pp. 1351-1363.
@article{50cc3f49db504033afe7ac457115c427,
title = "Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)",
abstract = "Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45 C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K m and V max were found to be 38.59 μM and 0.8237 μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037 μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.",
keywords = "Glycosyl hydrolase family 1, Homology modeling, Leucaena leucocephala, Molecular docking",
author = "Shaik, {Noor M.} and Anurag Misra and Somesh Singh and Fatangare, {Amol B.} and Suryanarayanarao Ramakumar and Rawal, {Shuban K.} and Khan, {Bashir M.}",
year = "2013",
month = "2",
doi = "10.1007/s11033-012-2179-6",
language = "English (US)",
volume = "40",
pages = "1351--1363",
journal = "Molecular Biology Reports",
issn = "0301-4851",
publisher = "Springer Netherlands",
number = "2",

}

TY - JOUR

T1 - Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

AU - Shaik, Noor M.

AU - Misra, Anurag

AU - Singh, Somesh

AU - Fatangare, Amol B.

AU - Ramakumar, Suryanarayanarao

AU - Rawal, Shuban K.

AU - Khan, Bashir M.

PY - 2013/2

Y1 - 2013/2

N2 - Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45 C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K m and V max were found to be 38.59 μM and 0.8237 μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037 μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

AB - Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45 C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K m and V max were found to be 38.59 μM and 0.8237 μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037 μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

KW - Glycosyl hydrolase family 1

KW - Homology modeling

KW - Leucaena leucocephala

KW - Molecular docking

UR - http://www.scopus.com/inward/record.url?scp=84878377537&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84878377537&partnerID=8YFLogxK

U2 - 10.1007/s11033-012-2179-6

DO - 10.1007/s11033-012-2179-6

M3 - Article

C2 - 23079707

AN - SCOPUS:84878377537

VL - 40

SP - 1351

EP - 1363

JO - Molecular Biology Reports

JF - Molecular Biology Reports

SN - 0301-4851

IS - 2

ER -