title: Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans.
abstract: BACKGROUND
  Transglycosylation (TG) activity is a property of glycosyl hydrolases (GHs) with which new glycosidic bonds are introduced between donor and acceptor sugar molecules. This special property of the GHs has potential to generate longer chain chitooligosaccharides (CHOS) that show elicitor activity in plants. We hypothesize that TG activity could be improved by retaining the substrate for a longer duration in the catalytic site.
  
  
  METHODS
  Four variants of chitinase D from Serratia proteamaculans (SpChiD) i.e. G119S, G119W, W120A and G201W were analyzed in detail for improved TG activity using high performance liquid chromatography (HPLC) and high resolution mass spectrometry (HRMS). The results were strongly supported by 50ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE).
  
  
  RESULTS
  The mutant G119W lost much of both hydrolytic and TG activities, while the mutant G201W displayed increased TG. The trajectory of MD simulations of the mutant G119W showed that the indole rings of two adjacent Trp residues create a major hindrance for the DP4 movement towards the catalytic center. Increased van der Waals (vdW) and coulombic interactions between DP4 substrate and the Trp-201 resulted in enhanced TG activity with the mutant G201W. The average number of hydrogen bonds observed for the DP4 substrate was increased for the mutants G119W and G201W compared to SpChiD.
  
  
  CONCLUSIONS
  The increase in TG activity could be due to partial blocking of product exit of SpChiD.
  
  
  CONCLUSIONS
  This new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.
authors: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India.
authors: Jogi
authors: J
authors: Madhuprakash
authors: School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India.
authors: Karunakar
authors: K
authors: Tanneeru
authors: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India.
authors: Bhavana
authors: B
authors: Karlapudi
authors: School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India. Electronic address: lgpsc@uohyd.ernet.in.
authors: Lalitha
authors: L
authors: Guruprasad
authors: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India. Electronic address: arpsl@uohyd.ernet.in.
authors: Appa Rao
authors: AR
authors: Podile
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012705:Serratia / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D002688:Chitinases
keywords: Chitinases; Chitooligosaccharides; Molecular Dynamics Simulations; Mutagenesis; SpChiD; Transglycosylation