Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse beta-carotene 15, 15'-monooxygenase.

ABSTRACT:

Background

beta-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a beta-carotene-accumulating E. coli system.

Results

Our assays show that mutation of either Y235 or Y326 to leucine (no cation-pi stabilization) significantly impair

SUBMITTER: Poliakov E 

PROVIDER: S-EPMC2801523 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature