Role of Glu445 in the substrate binding of beta-glucosidase

Manish Kumar Tiwari, Kyoung-Mi Lee, Dayanand Kalyani, Raushan Kumar Singh, Hoon Kim, Jung-Kul Lee, Priyadharshini Ramachandran

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a beta-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl beta-D-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k(cat)/K-m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested. (C) 2012 Elsevier Ltd. All rights reserved.
Original languageEnglish
JournalProcess Biochemistry
Volume47
Issue number12
Pages (from-to)2365-2372
ISSN1359-5113
DOIs
Publication statusPublished - 2012
Externally publishedYes

Keywords

  • BIOCHEMISTRY
  • BIOTECHNOLOGY
  • ENGINEERING,
  • GLYCOSYL-ENZYME INTERMEDIATE
  • BASIDIOMYCETE PHANEROCHAETE-CHRYSOSPORIUM
  • MOLECULAR RECOGNITION
  • CATALYTIC MACHINERY
  • THERMOPHILIC FUNGUS
  • STRUCTURAL-ANALYSIS
  • CRYSTAL-STRUCTURES
  • DRUG DESIGN
  • HYDROLASES
  • SPECIFICITY
  • beta-glucosidase
  • Molecular determinant
  • Molecular dynamics simulation
  • Density functional theory
  • Substrate binding
  • binding affinity
  • catalytic efficiency
  • crystal structure
  • density functional theory
  • GenBank sequence data
  • substrate affinity
  • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species host, expression system
  • Neosartorya fischeri BGL1 gene [Ascomycetes] Neosartorya fischeri beta-glucosidase gene aspartate, glutamic aci-445, substrate binding pocket, conserved amino acid, hydrogen bonding
  • carboxylate oxygen
  • glycoside hydrolase 9032-92-2 EC 3.2.2.20 substrate binding
  • para-nitrophenyl beta-dextro-glucopyranoside
  • substrate
  • 03502, Genetics - General
  • 03504, Genetics - Plant
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • 51518, Plant physiology - Enzymes
  • Biochemistry and Molecular Biophysics
  • homology modeling mathematical and computer techniques
  • molecular determination laboratory techniques
  • molecular dynamics simulation mathematical and computer techniques
  • sequence alignment laboratory techniques, mathematical and computer techniques, genetic techniques
  • site-directed mutagenesis laboratory techniques, genetic techniques
  • Enzymology
  • Molecular Genetics
  • GENE expression in Escherichia coli
  • ENZYMES
  • FUNGI
  • GLYCOSIDASES
  • KINETICS
  • NEOSARTORYA
  • RECOMBINANT ENZYMES
  • STRUCTURE
  • β-GLUCOSIDASES
  • Biotechnology
  • Enzyme systems
  • Amino acids
  • Binding energy
  • Carboxylation
  • Cloning
  • Escherichia coli
  • Hydrogen bonds
  • Molecular dynamics
  • Substrates
  • β-glucosidase
  • Acidic amino acids
  • Aspartates
  • Binding affinities
  • Catalytic efficiencies
  • Density functional theories (DFT)
  • Glucosidase
  • Homology modeling
  • Molecular determinants
  • Molecular dynamics simulations
  • Neosartorya fischeri
  • Sequence alignments
  • Site directed mutagenesis
  • Substrate affinity
  • Substrate recognition
  • Wild types

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