Deciphering the Myrosinase-like Activity of Lactiplantibacillus plantarum WCFS1 among GH1 Family Glycoside Hydrolases

Abstract

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-β-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic β-thio-glucoside derivative or against the β-glucoside. However, all 11 proteins were active on the phosphorylated-β-glucoside derivative. Moreover, only eight of these proteins were active on phospho-β-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates.

Dual 6Pβ-Galactosidase/6Pβ-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1

“…These 11 GH1 proteins have been recently characterized. All of them presented 6Pβ-glc activity, and in addition, 8 proteins exhibited 6-phospho-β- d -thioglucosidase activity . Therefore, it is possible that some of these GH1 proteins could also possess 6Pβ-gal activity, being responsible for the 6Pβ-gal activity exhibited by L.…”

“…The 11 GH1 6Pβ-glc proteins and LacA β-gal were recombinantly produced as previously described. , The hydrolytic activity of these enzymes was assayed by using p NP-β- d -galactopyranoside ( p NP-Gal) and p NP-6-phospho-β- d -galactopyranoside ( p NP-6P-Gal) as substrates, for β-gal and 6Pβ-gal activities, respectively. LacA GH42 proteins were used as the control for β-gal activity .…”

“…13 In addition, the catalytic activity of these proteins toward 24 pNP glucoside derivatives was also assayed. Despite all the GH1 proteins exhibiting 6Pβglc activity, 20 only one of them, Lp_3525 (Pbg9), effectively hydrolyzed pNP-6P-Gal and showed high 6Pβ-gal activity (Table 1). Low 6Pβ-gal activity was observed in Lp_1401, Lp_2777, and Lp_2778 proteins.…”

“…20 Thus, the catalytic acid/base and nucleophilic Glu residues (Glu-181 and Glu-378), the residues involved in glycon binding (−1 subsite), and those involved in aglycon binding (+1 subsite) are conserved. 20 Moreover, the Lp_3525 sequence presents a long extra C-terminal segment unique to 6Pβ-glc, which varies in length and sequence. This is a typical 6Pβ-glc motif that is absent in the 6Pβ-gal hydrolases.…”

“…Glycoside hydrolases from the GH1 family (Lp_0440, Lp_0906, Lp_1401, Lp_2777, Lp_2778, Lp_3011, Lp_3132, Lp_3512, Lp_3525, Lp_3526, and Lp_3629 proteins) and GH42 family (LacA or Lp_3469) from L. plantarum WCFS1 were produced as previously described. , The hydrolytic activity of these 12 glycoside hydrolases was determined by using a library of 24 p NP-glycoside derivatives . The assay was performed in a 96-well flat bottom plate (Sarstedt), where each well contained a different substrate (10 mM).…”