Abstract
Among α-transglucosylases from Glycoside-Hydrolase family 70, the ΔN123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1μs) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ΔN123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of ΔN123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.
Highlights
Sucrose-utilizing α-transglucosylases from Glycoside-Hydrolase family 70 (GH70) [1], are extracellular bacterial enzymes of high molecular weight, typically in the range of 120–300 kDa, that naturally synthesize from sucrose substrate a wide variety of linear and branched αglucan polymers differing in terms of type of glucosidic linkages, degree and spatial arrangement of branches, molecular size and physico-chemical properties [2,3,4]
We report here for the first time the computational investigation of one member of GH70 family, the branching sucrase ΔN123-glucan-binding domain (GBD)-CD2, based on a combination of large scale molecular dynamics (MD) simulations at the microsecond time range, Normal Mode Analysis (NMA) and Essential Dynamics Analysis (EDA)
Considering the μs scale of the MD simulation on ΔN123-GBD-CD2, the Kullback-Leibler Divergence (KLD) appeared more appropriate to quantify the convergence as a function of the simulation time using the Principal Component histograms corresponding respectively to the first or the second half of the trajectories (1 to 500ns vs 500ns to 1μs)
Summary
Sucrose-utilizing α-transglucosylases from Glycoside-Hydrolase family 70 (GH70) [1], are extracellular bacterial enzymes of high molecular weight, typically in the range of 120–300 kDa, that naturally synthesize from sucrose substrate a wide variety of linear and branched αglucan polymers differing in terms of type of glucosidic linkages, degree and spatial arrangement of branches, molecular size and physico-chemical properties [2,3,4]. The so-called dextransucrases (DSR), catalyze the formation of dextrans composed of a linear chain mainly formed of α-1,6 linked glucans on which are branched glucosyl chains through various types of α-1,2, α-1,3, and α-1,4 linkages [4] One of these enzymes, DSR-E from L. citreum NRRL B-1299 [6], has been extensively studied in our group, revealing very original catalytic properties as it is able to synthesize dextran with high amounts of rare and non-digestible α-1,2 branching linkages. Based on its unique properties, GBD-CD2 was reported as being the first engineered Branching Sucrase (BRS), not able to elongate glucan polymers [6,8] These results paved the way for the discovery through data mining of novel natural GH70 branching sucrases specialized in dextran branching via α-1,2 or α-1,3 osidic linkages [9,10,11]. The α-1,3 branched glucan shows interesting properties, especially in the inhibition of bacteria such as Salmonella or Escherichia coli [17]
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