Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) β-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil

Abstract

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm–3 min–1 mg–1, respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67

Changes in rocket salad phytochemicals within the commercial supply chain: Glucosinolates, isothiocyanates, amino acids and bacterial load increase significantly after processing

“…It has been documented that soil bacteria ( Citrobacter spp.) possess a glucoside hydrolase family 3 (GH3) β-glucosidase enzyme, which may potentially aid them in the scavenging of glucose from GSLs (Albaser et al, 2016). The same may be true of bacteria that live on leaves, but very little research has been conducted in this area.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Generally β-glucosidases belonging to GH 1 have 400-550 amino acids in length with molecular weight ranges 40-60 kDa [64,156]. Similarly, β-glucosidases belonging to GH family 3 contain 600-900 amino acids in length with molecular weight of 65-90 kDa per subunit, but because these group of enzymes are usually glycosylated, their molecular mass ranges 110-130 kDa [44,157,158]. β-Glucosidase varies in quaternary structure arrangement, for example, monomeric [159], dimeric [160], trimeric [147], tetrameric [151] enzymes have been reported.…”

Gut Glucosinolate Metabolism and Isothiocyanate Production

“…For bacterial myrosinases, little is known concerning the structure of myrosinase with the exception of Citrobacter Wye1 where a complete gene sequence has been identified . This sequence was based on the actual genome of Citrobacter Wye1 together with an N‐terminal sequence and peptide sequences from tryptic digests of the purified myrosinase.…”

“…A feature of the GH3 β‐O‐glucosidase is the signature “SDW” conserved motif as is the case for Citrobacter Wye1 myrosinase and contains aspartate as the catalytic nucleophile rather than glutamate that is characteristic of GH1 plant myrosinases. There was very little homology between the Citrobacter Wye1 and plant or aphid myrosinases . If a 6‐phospho‐β‐glucosidase is responsible for the metabolism of sinigrin by E. coli 0157:H7 then it is possible that the GSL substrate requires phosphorylation at the 6‐hydroxyl position on the glucose residue of the GSL ( Figure ).…”