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 biosynthe tic pathway of Rhodobacter species. The gene encoding this enzyme was expressed in Escherichia coli, purified, and biochemically characteriz ed. The resulting protein contained an N-terminal six-histidine extens ion which derived from the cloning vector; this allowed for a one-step purification of the enzyme to homogeneity after solubilization with N onidet P-40. The hydrogen acceptor in the C-3,4 desaturation reaction aas molecular oxygen. NAD(+), NADP(+), and flavin adenine dinucleotide had no influence on enzymatic activity. Different acyclic 1-hydroxyca rotenoids were tested as substrates. Very good conversion was achieved with 1-hydroxyneurosporene and 1-hydroxylycopene, whereas 1-hydroxy-g amma-carotene and 1,1'-dihydroxylycopene were much less effective. Fro m 1'-hydroxy-3,4-didehydrolycopene only trace amounts of product were obtained, and 1-methoxyneurosporene was not converted by purified hydr oxyneurosporene desaturase. A K-m of 13.4 mu M was determined for 1-hy droxyneurosporene.