Expression, purification and properties of lycopene cyclase from Erwinia uredovora.
Lycopene cyclase, an enzyme responsible for the formation of cyclic carotenoids from acyclic precursors has been purified to homogeneity in an active state. The Erwinia uredovora lycopene cyclase gene (crtY) was over-expressed in Escherichia coli. From this recombinant strain the enzyme was purified by immuno-affinity chromatography and its cyclization activity characterized as a two-step reaction in which both sides of the lycopene molecule are cyclized to beta-ionone rings with the monocyclic gamma-carotene as an intermediate. Furthermore, neurosporene as well as l-hydroxylycopene were cyclized to beta-zeacarotene and hydroxy-gamma-carotene respectively. In contrast, neither 1,1'- dihydroxylycopene nor the tetra-cis-prolycopene were accepted as substrates. The cofactors involved in the reaction were either NADH or NADPH. K(m) values were determined for lycopene and NADPH to be 1.8 microM and 2.5 mM respectively.
Schnurr
G
G
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Federal Republic of Germany.
Misawa
N
N
Sandmann
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002338:Carotenoids / Q000096:biosynthesis / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004885:Erwinia / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D019753:Intramolecular Lyases; D007535:Isomerases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009243:NAD; D002338:Carotenoids; D009249:NADP; D007535:Isomerases; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase