Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase |
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Authors: | Eugenia Poliakov Susan Gentleman Preethi Chander Francis X Cunningham Jr Bella L Grigorenko Alexander V Nemuhin T Michael Redmond |
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Affiliation: | (1) National Eye Institute, NIH, Bethesda, MD 20892-0608, USA;(2) Department of Cell Biology and Molecular Genetics, University of Maryland,College Park, Maryland, 20742, USA;(3) Chemistry Department, MV Lomonosov Moscow State University, Moscow, Russia |
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Abstract: | Background β-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 β-carotene-accumulating E. coli system. |
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