Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum

Psilotum nudum Griseb is a pteridophyte and belongs to the single family (P silotaceae) of the division, Psilophyta. Being the only living species of a once populated division, P. nudum is the most primitive vascular plant. Ch alcone synthase (CHS; EC 2.3.1.74) superfamily enzymes are responsible for biosyntheses of diverse secondary metabolites, including flavonoids and sti lbenes. Using a reverse transcription-polymerase chain reaction strategy, f our CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P. nud um, and heterologously expressed in Escherichia coli. These four enzymes of 396-406 amino acids showed sequence identity of > 50% among themselves and to other higher-plant CHS-superfamily enzymes. PnJ and PnP preferred p-cou maroyl-CoA and isovaleryl-CoA, respectively, as starter CoA and catalyzed C HS-type ring formation, indicating that they are CHS and phlorisovalerophen one synthase, respectively. On the other hand, PnI and PnL preferred cinnam oyl-CoA as starter CoA and catalyzed stilbene synthase-type cyclization and thus were determined to be pinosylvin synthases (EC 2.3.1.146). In additio n, PnE, which uniquely contains a glutamine in place of otherwise strictly conserved histidine, had no apparent in vitro catalytic activity. Phylogene tic analysis indicated that these P. nudum clones form a separate cluster t ogether with Equisetum arvense CHS. This cluster of pteridophytes is locate d next to the cluster formed by pine (gymnosperm) enzymes, in agreement wit h their evolutionary relationships. Psilotum nudum represents a plant with the most diverse CHS-superfamily enzymes and this ability to diverge may ha ve provided a survival edge during evolution.