Partial Purification and Characterization of a Monoterpene Cyclase, Limonene Synthase, from the LiverwortRicciocarpos natans

Abstract

4S-(−)-limonene is the major monoterpene accumulated by the liverwortRicciocarpos natans,and a cell-free extract from this nonvascular plant culturedin vitrocatalyzes the cyclization of geranyl diphosphate to limonene. The time course of limonene synthase activity parallels cultured growth but shows a maximum in specific activity in the lag phase following transfer to fresh medium. The operationally soluble enzyme was partially purified by combination of anion-exchange and hydroxylapatite chromatography. The limonene synthase fromR. natanspossesses a molecular weight of about 51,000, exhibits a pH optimum at 6.5, a pIat 5.3, and a requirement for either Mg2+or Mn2+as cofactor, and appears to employ 3S-linalyl diphosphate as a bound intermediate in the cyclization of the geranyl substrate (Km∼1.25 μM). In stereochemistry of the coupled isomerization–cyclization reaction and in general properties, the limonene synthase from this bryophyte resembles the corresponding monoterpene cyclases from gymnosperm and angiosperm species.