A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa

Phytoene desaturase Al-l from Neurospora crassa was expressed in Escherichi a coil and an active enzyme was isolated which catalyzed the stepwise intro duction of up to five double bonds into the substrate phytoene, The major r eaction products were 3,4-didehydrolycopene and lycopene. Several of the de saturation intermediates, zeta -carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-l, In contrast to the structurally relat ed bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-l. In situ competition with a neurosporene- and lycopene-co nverting hydratase and cyclase indicated that these enzymes can divert inte rmediates of the desaturation sequence. Based on the in vitro and in vivo r esults, the organization of the phytoene desaturase from N. crassa was prop osed as an assembly of identical protein units which are responsible for th e multistep reaction. However, the spatial arrangement should be loose enou gh to allow an exchange of individual intermediates in both directions in a nd out of this complex. Since gamma -carotene is not accepted as a substrat e by Al-l, the formation of torulene must proceed exclusively by the cycliz ation of 3,4-didehydrolycopene. (C) 2000 Academic Press.