Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse beta-carotene 15, 15'-monooxygenase.
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 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 beta-carotene mounted supports a mechanism involving cation-pi stabilization by Y235 and Y326.


CONCLUSIONS
Our data are consistent with the formation of a substrate carbocation intermediate and cation-pi stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.
Poliakov
Eugenia
E
National Eye Institute, NIH, Bethesda, MD 20892-0608, USA. poliakove@nei.nih.gov
Gentleman
Susan
S
Chander
Preethi
P
Cunningham
Francis X
FX
Grigorenko
Bella L
BL
Nemuhin
Alexander V
AV
Redmond
T Michael
TM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002412:Cations / Q000737:chemistry*; D004159:Diphenylamine / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism; D019207:beta Carotene / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002412:Cations; D019207:beta Carotene; D014443:Tyrosine; D004159:Diphenylamine; D050559:beta-Carotene 15,15'-Monooxygenase