Quantum mechanics/molecular mechanics modeling of substrate-assisted catalysis in family 18 chitinases: conformational changes and the role of Asp142 in catalysis in ChiB

Jitrayut Jitonnom, Vannajan S Lee, Piyarat Nimmanpipug, Heather A Rowlands, Adrian J Mulholland

Research output: Contribution to journalArticle (Academic Journal)peer-review

46 Citations (Scopus)

Abstract

Family 18 chitinases catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin. The mechanism has been proposed to involve the formation of an oxazolinium ion intermediate via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile (instead of an enzyme residue). Here, we have modeled the first step of the chitin hydrolysis catalyzed by Serratia marcescens chitinase B for the first time using a combined quantum mechanics/molecular mechanics approach. The calculated reaction barriers based on multiple snapshots are 15.8-19.8 kcal mol(-1) [B3LYP/6-31+G(d)//AM1-CHARMM22], in good agreement with the activation free energy of 16.1 kcal mol(-1) derived from experiment. The enzyme significantly stabilizes the oxazolinium intermediate. Two stable conformations ((4)C(1)-chair and B(3,O)-boat) of the oxazolinium ion intermediate in subsite -1 were unexpectedly observed. The transition state structure has significant oxacarbenium ion-like character. The glycosyl residue in subsite -1 was found to follow a complex conformational pathway during the reaction ((1,4)B → [(4)H(5)/(4)E](++) → (4)C(1) ↔ B(3,O)), indicating complex conformational behavior in glycoside hydrolases that utilize a substrate-assisted catalytic mechanism. The D142N mutant is found to follow the same wild-type-like mechanism: the calculated barriers for reaction in this mutant (16.0-21.1 kcal mol(-1)) are higher than in the wild type, in agreement with the experiment. Asp142 is found to be important in transition state and intermediate stabilization.

Original languageEnglish
Pages (from-to)4697-711
Number of pages15
JournalBiochemistry
Volume50
Issue number21
DOIs
Publication statusPublished - 31 May 2011

Keywords

  • Aspartic Acid
  • Biocatalysis
  • Chitinase
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Protein Conformation
  • Quantum Theory

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