Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity

Abstract

β-Fructosidase, a glycoside hydrolase of a biotechnologically important strain, was studied for its biochemical, physicochemical, and three-dimensional structure characteristics. This enzyme was heterologously expressed in Escherichia coli as a C-terminal His-tagged protein (SacB). β-Fructosidase catalyzes the cleavage of glycoside bonds toward certain carbohydrates with β-fructofuranosyl linkages; however, SacB exhibited selectivity toward sucrose and an optimum activity at pH 6.0-6.5 and 37 °C. In such optimum enzymatic activity conditions, the SacB was commonly observed as a monodisperse protein by dynamic light scattering (DLS). As β-fructosidase belongs to glycoside hydrolase family 32 (GH32), a β-sandwich and a five-bladed β-propeller domain are typical predicted folds in its structure. Docking and molecular dynamic simulations revealed for the first time a funnel-like channel perfectly exposed in the β-propeller domain of the Lactobacillus plantarum β-fructosidase (this allows the interaction between its entire catalytic triad and substrates that are larger than sucrose). In contrast, SacB showed a closed central tunnel collaterally induced by its His-tag.

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other β-Fructofuranosidases in Bacteria

“…The pH optimum of each enzyme is relatively high compared to other intracellular β-fructofuranosidases, which are usually between 5.5 and 6.5 for both Suc6P hydrolases and invertases. ,,,,,, For instance, SacA from B. subtilis has an optimal pH of 6.5, while the optimal pH of SacAP1 within this study has been determined at 7.2 .…”

“…104 The pH optimum of each enzyme is relatively high compared to other intracellular β-fructofuranosidases, which are usually between 5.5 and 6.5 for both Suc6P hydrolases and invertases. 7,27,69,70,74,103,105 For instance, SacA from B. subtilis has an optimal pH of 6.5, while the optimal pH of SacAP1 within this study has been determined at 7.2. 7 Intracellular pH of growing cells of P. megaterium has been reported to be rather alkaline, around pH 8.1, which is similar to what has been reported for B. subtilis under exponential growth (pH 8.0).…”

“…7,26,27,102 Other characterized GH32 enzymes grouping into the intracellular PTS enzymes also have high K m values for Suc: 200 mM and 66 mM for LgAs32 from L. gasseri and β-fructosidase from L. plantarum, respectively. 74,103 For them, the K m for Suc6P is unknown. Previous mutagenesis experiments showed that the K m for Suc of an invertase from plant origin (Arabidopsis cell wall Invertase1) increases 200-fold when the tryptophan of the Suc-binding box is changed into a leucine (which is the case for SacAP1 and the majority of Suc6P hydrolases).…”

“…This may also be the reason why Mendoza-Llereneas et al (2016) did not find affinity of Suc6P hydrolase from L. plantarum toward IOS, as they did not take into account a potential role of phosphorylation. 103 Moreover, the positively charged residues found in Suc6P hydrolases may inhibit the binding of nonphosphorylated sugars. Regardless of these complexities, our work helps to better discriminate between putative Suc6P hydrolases, invertases, and fructanases from the GH32 family, derived from freshly sequenced bacterial genomes.…”

Okara: A Nutritionally Valuable By-product Able to Stabilize Lactobacillus plantarum during Freeze-drying, Spray-drying, and Storage

“…The addition of sucrose in the dehydration medium improved the recovery of microorganisms grown in defatted okara and in MRS ( Figure 3 ), which was an expected result considering the reported protective effect of sucrose during preservation of lactic acid bacteria (Tymczyszyn et al, 2007a,b). In this regard, it is worth to mention that although there are recent articles reporting the sucrose metabolism in L. plantarum strains (Chen et al, 2014; Mendoza-Llerenas et al, 2016), in this work, L. plantarum CIDCA 83114 is exposed to sucrose in non-growing conditions. Thus, sucrose is not hydrolyzed and acts as a protective agent.…”