Kinetic Characterization of Prenyl-Flavin Synthase from &ITSaccharomyces cerevisiae&IT
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作者:
Arunrattanamook, Nattapol
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Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Chem Engn, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Arunrattanamook, Nattapol
[1
,2
]
Marsh, E. Neil G.
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Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Marsh, E. Neil G.
[1
,3
]
机构:
[1] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Dept Chem Engn, Ann Arbor, MI 48109 USA
[3] Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USA
We have characterized the kinetics and substrate requirements of prenyl-flavin synthase from yeast. This enzyme catalyzes the addition of an isopentenyl unit to reduced flavin mononucleotide (FMN) to form an additional six-membered ring that bridges NS and C6 of the flavin nucleus, thereby converting the flavin from a redox cofactor to one that supports the decarboxylation of aryl carboxylic acids. In contrast to bacterial enzymes, the yeast enzyme was found to use dimethylallyl pyrophosphate, rather than dimethylallyl phosphate, as the prenyl donor in the reaction. We developed a coupled assay for prenylflavin synthase activity in which turnover was linked to the activation of the prenyl-flavin-dependent enzyme, ferulic acid decarboxylase. The kinetics of the reaction are extremely slow: k(cat) = 12.2 +/- 0.2 11 h(-1), and K-M for dimethylallyl pyrophosphate = 9.8 +/- 0.7 mu M. The K-M for reduced FMN was too accurately measured. The kinetics of reduced FMN consumption were studied under pre-steady state conditions. The reaction of FMN was described well by first-order kinetics with a k(app) of 17.4 +/- 1.1 h(-1). These results indicate that a chemical step, most likely formation of the carbon-carbon bond between C6 of the flavin and the isopentenyl moiety, is substantially rate-determining in the reaction.
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ferguson, Kyle L.
;
Eschweiler, Joseph D.
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Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Eschweiler, Joseph D.
;
Ruotolo, Brandon T.
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Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ruotolo, Brandon T.
;
Marsh, E. Neil G.
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机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ferguson, Kyle L.
;
Arunrattanamook, Nattapol
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机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Chem Engn, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Arunrattanamook, Nattapol
;
Marsh, E. Neil G.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ferguson, Kyle L.
;
Eschweiler, Joseph D.
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机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Eschweiler, Joseph D.
;
Ruotolo, Brandon T.
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机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ruotolo, Brandon T.
;
Marsh, E. Neil G.
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h-index: 0
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Ferguson, Kyle L.
;
Arunrattanamook, Nattapol
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机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Chem Engn, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Arunrattanamook, Nattapol
;
Marsh, E. Neil G.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USAUniv Michigan, Dept Chem, Ann Arbor, MI 48109 USA