TY - JOUR
T1 - Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH
AU - Yeh, Ellen
AU - Cole, Lindsay J.
AU - Barr, Eric W.
AU - Bollinger, J. Martin
AU - Ballou, David P.
AU - Walsh, Christopher T.
PY - 2006/6/27
Y1 - 2006/6/27
N2 - The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH 2 (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH2 and O2 in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH2 and O 2 was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s-1, quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s-1 at 25 °C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH2, chloride ion, and O2.
AB - The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH 2 (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH2 and O2 in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH2 and O 2 was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s-1, quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s-1 at 25 °C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH2, chloride ion, and O2.
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U2 - 10.1021/bi060607d
DO - 10.1021/bi060607d
M3 - Article
C2 - 16784243
AN - SCOPUS:33745963527
SN - 0006-2960
VL - 45
SP - 7904
EP - 7912
JO - Biochemistry
JF - Biochemistry
IS - 25
ER -