Characterized enzymes in the Phytoene synthase like subgroup are phytoene synthases, though they may vary in the stereochemistry of the phytoene product (EC 2.5.1.99 vs 2.5.1.32). In a subset of these enzymes, found in various fungal species, the phytoene synthase is part of a multidomain enzyme that also includes lycopene cyclase (not a member of the IS I superfamily). Most, however, are single domain enzymes. In addition to phytoene synthase, this subgroup may contain members that catalyze additional reactions that have not yet been characterized.
Velayos A, Eslava AP, Iturriaga EA
A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides
▸ Abstract
Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M. circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi. This gene was identified using complementation tests among different classes of carotenoid mutants of M. circinelloides. The carRP gene product contains two domains: the R domain is located at the N-terminus and determines lycopene cyclase activity; the P domain is located at the C-terminus and displays phytoene synthase activity. The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function. The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp. Northern analyses show a co-ordinated regulation of the expression of both genes by blue light. Several motifs found in this promoter region suggest a bi-directional mode of transcription control.
Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase
▸ Abstract
Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene.
