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Engineering a branching sucrase for flavonoid glucoside diversification.

ABSTRACT: Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an ?-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mutations inferred by molecular docking simulations were generated and screened for quercetin glucosylation using sucrose as a glucosyl donor. Only a limited number of variants (22) were retained on the basis of quercetin conversion and product profile. Their acceptor promiscuity towards five other flavonoids was subsequently assessed, and the automated screening effort revealed variants showing remarkable ability for luteolin, morin and naringenin glucosylation with conversion ranging from 30% to 90%. Notably, naringenin and morin, a priori considered as recalcitrant compounds to glucosylation using this ?-transglucosylases, could also be modified. The approach reveals the potential of small platforms of engineered GH70 ?-transglucosylases and opens up the diversity of flavonoid glucosides to molecular structures inaccessible yet.

SUBMITTER: Malbert Y

PROVIDER: S-EPMC6181985 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Engineering a branching sucrase for flavonoid glucoside diversification.

Malbert Yannick Y Moulis Claire C Brison Yoann Y Morel Sandrine S André Isabelle I Remaud-Simeon Magali M

Scientific reports 20181011 1

Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an α-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mu ...[more]

PMID: 30310109

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Project description:Previously, we have identified and characterized 4,6-?-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (?1?4)-linkages in amylose and introduce (?1?6)-linkages in linear chains. The 4,6-?-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (?1?6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1?2,6)-?-d-Glcp-(1?6)- (Leuconostoc citreum enzyme) or -(1?3,6)-?-d-Glcp-(1?6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1?3,6)-?-d-Glcp-(1?6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (?1?3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various ?-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (?1?3)-linkages present. The treatment of amylose with 4,6-?-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived ?-glucans with markedly reduced digestibility. These starch derived ?-glucans may find applications in the food industry.

Project description:Among ?-transglucosylases from Glycoside-Hydrolase family 70, the ?N123-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 (1?s) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ?N123-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 ?N123-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.

Dataset Information

Engineering a branching sucrase for flavonoid glucoside diversification.

Publications

Engineering a branching sucrase for flavonoid glucoside diversification.

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