کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1947586 1054618 2014 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans
چکیده انگلیسی


• We analyzed four variants of SpChiD for improved TG activity using HPLC and HRMS.
• MD simulations performed for SpChiD and its 4 mutants in complex with DP4 substrate
• G119W lost hydrolysis and TG to a greater extent but G201W displayed increased TG.
• A new approach to increase the TG by partial blocking of product exit in SpChiD

BackgroundTransglycosylation (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.MethodsFour 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 50 ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE).ResultsThe 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.ConclusionThe increase in TG activity could be due to partial blocking of product exit of SpChiD.General significanceThis new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - General Subjects - Volume 1840, Issue 9, September 2014, Pages 2685–2694
نویسندگان
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