Insight into naphthoquinone metabolism: β-glucosidase-catalysed hydrolysis of hydrojuglone β-d-glucopyranoside

Abstract

In plants, the naphthoquinone juglone is known to be involved in pathogenic defence mechanisms, but it may also take part in plant developmental processes. This naphthoquinone can accumulate in a glycosylated form, namely hydrojuglone β-d-glucopyranoside. The structural configuration of this compound was shown to be 1,5-dihydroxy-4-naphthalenyl-β-d-glucopyranoside by means of MS, NMR and nuclear Overhauser effect spectroscopy analyses. A hydrojuglone β-d-glucopyranoside β-glucosidase (EC 3.2.1.21) was purified to homogeneity from Juglans regia L. The enzyme catalysed the release of juglone from hydrojuglone β-d-glucopyranoside with high specificity and showed Michaelis–Menten kinetics with Km = 0.62 mM and Vmax = 14.5 µkat/mg of protein. This enzyme also showed a higher activity towards β-d-fucosyl than β-d-glucosyl bonds. The purified enzyme had an apparent Mr of 64000 by SDS/PAGE and a pI 8.9 by isoelectrofocusing PAGE. The purified enzyme was inhibited by several bivalent cations, such as Cu2+, Fe2+, Hg2+, and by d-glucono-1,5-lactone, showing non-competitive inhibition of the mixed type.