Characteristics and molecular determinants of a highly selective and efficient glycyrrhizin-hydrolyzing β-glucuronidase from Staphylococcus pasteuri 3I10
Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-β-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 β-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified β-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 °C, respectively. Comparing to E. coli β-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (Vmax 16.1 ± 0.9 vs 140.0 ± 4.1 μmolmin−1 mg−1; Km 469.4 ± 73.4 vs 268.0 ± 25.8 μM), but could selectively convert GL to GA at much higher efficiency (Vmax 0.41 ± 0.011 vs 0.005 ± 0.002 μmolmin−1 mg−1; Km 116.9 ± 15.4 vs 53.4 ± 34.8 μM). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 β-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 β-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 β-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.
KeywordsStaphylococcus pasteuri Bacterial β-glucuronidase Glycyrrhizin Glycyrrhetinic acid Deglucuronidation Homology modeling
This work was financially supported by the National Natural Science Foundation (Ref. no: 81473281), University of Macau (MYRG2015-00220-ICMS-QRCM)), and the Science and Technology Development fund of Macao SAR (043/2011/A2, 029/2015/A1).
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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