Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides.
Hydroxyneurosporene desaturase is involved in the carotenoid biosynthetic pathway of Rhodobacter species. The gene encoding this enzyme was expressed in Escherichia coli, purified, and biochemically characterized. The resulting protein contained an N-terminal six-histidine extension which derived from the cloning vector; this allowed for a one-step purification of the enzyme to homogeneity after solubilization with Nonidet P-40. The hydrogen acceptor in the C-3,4 desaturation reaction was molecular oxygen. NAD+, NADP+, and flavin adenine dinucleotide had no influence on enzymatic activity. Different acyclic 1-hydroxycarotenoids were tested as substrates. Very good conversion was achieved with 1-hydroxyneurosporene and 1-hydroxylycopene, whereas 1-hydroxy-gamma-carotene and 1,1'-dihydroxylycopene were much less effective. From 1'-hydroxy-3,4-didehydrolycopene only trace amounts of product were obtained, and 1-methoxyneurosporene was not converted by purified hydroxyneurosporene desaturase. A Km of 13.4 microM was determined for 1-hydroxyneurosporene.
Albrecht
M
M
Biosynthesis Group, Botanical Institute, J.W. Goethe Universität, Frankfurt, Germany.
Ruther
A
A
Sandmann
G
G
D002338:Carotenoids / Q000096:biosynthesis*; D003067:Coenzymes; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013379:Substrate Specificity
D003067:Coenzymes; D011994:Recombinant Proteins; C005637:spheroidene; D002338:Carotenoids; D010088:Oxidoreductases; C075277:methoxyneurosporene dehydrogenase; C109407:hydroxyneurosporene desaturase