Biochemical characterization of purified zeta-carotene desaturase from Anabaena PCC 7120 after expression in Escherichia coli.
A novel enzyme, zeta-carotene desaturase from the cyanobacterium Anabaena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowed the purification of this enzyme and subsequent enzyme kinetic studies. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion-exchange and hydrophobic interaction chromatography. The correct translational start was confirmed by N-terminal protein sequencing. Substrates for zeta-carotene desaturase apart from zeta-carotene are those carotenes which partially resemble the latter, like neurosporene and beta-zeacarotene yielding lycopene and gamma-carotene, respectively as reaction products. Also cis isomers like pro-zeta-carotene were converted to the corresponding products. Km values of 10 microM were determined for both substrates zeta-carotene and neurosporene. The enzyme was inhibited to some extent by the experimental herbicides J852 and LS80707 and also by diphenylamine which is a well-known inhibitor of the bacterial-type phytoene desaturase.
Albrecht
M
M
Botanisches Institut, J.W.Goethe-Universität, Frankfurt, Germany.
Linden
H
H
Sandmann
G
G
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism*; D002793:Cholic Acids; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; D011994:Recombinant Proteins; D002338:Carotenoids; D010088:Oxidoreductases; C099215:zeta-carotene desaturase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate