PARTIAL-PURIFICATION AND CHARACTERIZATION OF A MONOTERPENE CYCLASE, LIMONENE SYNTHASE, FROM THE LIVERWORT RICCIOCARPOS NATANS

4S-(-)-limonene is the major monoterpene accumulated by the liverwort Ricciocarpos natans, and a cell-free extract from this nonvascular pla nt cultured in vitro catalyzes the cyclization of geranyl diphosphate to limonene. The time course of limonene synthase activity parallels c ultured growth but shows a maximum in specific activity in the lag pha se following transfer to fresh medium. The operationally soluble enzym e was partially purified by combination of anion-exchange and hydroxyl apatite chromatography. The limonene synthase from R. natans possesses a molecular weight of about 51,000, exhibits a pH optimum at 6.5, a p I at 5.3, and a requirement for either Mg2+ or Mn2+ as cofactor, and a ppears to employ 3S-linalyl diphosphate as a bound intermediate in the cyclization of the geranyl substrate (K-m similar to 1.25 mu M). In s tereochemistry of the coupled isomerization-cyclization reaction and i n general properties, the limonene synthase from this bryophyte resemb les the corresponding monoterpene cyclases from gymnosperm and angiosp erm species. (C) 1996 Academic Press, Inc.