Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase.
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.
Iwata-Reuyl
Dirk
D
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Math
Shivanand K
SK
Desai
Shrivallabh B
SB
Poulter
C Dale
CD
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004885:Erwinia / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase