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

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dc.contributor.authorPoliakov, Eugenia
dc.contributor.authorGentleman, Susan
dc.contributor.authorChander, Preethi
dc.contributor.authorCunningham, Francis X Jr
dc.contributor.authorGrigorenko, Bella L
dc.contributor.authorNemuhin, Alexander V
dc.contributor.authorRedmond, T Michael
dc.date.accessioned2021-11-18T17:29:58Z
dc.date.available2021-11-18T17:29:58Z
dc.date.issued2009-12-14
dc.description.abstractβ-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. Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326. Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.en_US
dc.description.urihttps://doi.org/10.1186/1471-2091-10-31
dc.identifierhttps://doi.org/10.13016/4kqa-iaao
dc.identifier.citationPoliakov, E., Gentleman, S., Chander, P. et al. Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase. BMC Biochem 10, 31 (2009).en_US
dc.identifier.urihttp://hdl.handle.net/1903/28142
dc.language.isoen_USen_US
dc.publisherSpringer Natureen_US
dc.relation.isAvailableAtCell Biology & Molecular Geneticsen_us
dc.relation.isAvailableAtDigital Repository at the University of Marylanden_us
dc.relation.isAvailableAtCollege of Computer, Mathematical & Natural Sciencesen_us
dc.relation.isAvailableAtUniversity of Maryland (College Park, MD)en_us
dc.subjectCarotenoiden_US
dc.subjectAromatic Residueen_US
dc.subjectDiphenylamineen_US
dc.subjectPolyene Chainen_US
dc.subjectRetinyl Palmitateen_US
dc.titleBiochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenaseen_US
dc.typeArticleen_US

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