Mechanistic Studies on CymD: A Tryptophan Reverse N-Prenyltransferase

被引:21
作者
Qian, Qi [1 ]
Schultz, Andrew W. [2 ,3 ]
Moore, Bradley S. [2 ,3 ]
Tanner, Martin E. [1 ]
机构
[1] Univ British Columbia, Dept Chem, Vancouver, BC V6T 1Z1, Canada
[2] Univ Calif San Diego, Scripps Inst Oceanog, La Jolla, CA 92093 USA
[3] Univ Calif San Diego, Skaggs Sch Pharm & Pharmaceut Sci, La Jolla, CA 92093 USA
基金
美国国家卫生研究院; 加拿大自然科学与工程研究理事会;
关键词
INDOLE-DERIVATIVES; BIOSYNTHESIS; REARRANGEMENT; SYNTHETASE; ALKALOIDS; SYNTHASE; GERANYL;
D O I
10.1021/bi3009054
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The prenyltransferase CymD catalyzes the reverse N-prenylation of tryptophan using dimethylallyl diphosphate (DMAPP) in the biosynthesis of the cyclic peptides cyclomarin and cyclomarazine. The mechanism is of interest because a non-nucleophilic indole nitrogen must be alkylated in this process. Three mechanisms were initially considered, including (A) a direct addition of a carbocation to the nitrogen, (B) an addition of a carbocation to C-3 followed by an aza-Cope rearrangement, and (C) deprotonation of the indole followed by an S(N)2' addition to DMAPP. The use of 4-fluorotryptophan and 6-fluorotryptophan revealed that the reaction kinetics are only modestly affected by these substitutions, consistent with the notion that positive charge does not accumulate on the indole ring during catalysis. When (E)-3-(fluoromethyl)-2-buten-1-yl diphosphate was used in place of DMAPP, the maximal rate was reduced by a factor of 100, consistent with the development of positive charge on the dimethylallyl moiety. Positional isotope exchange (PIX) experiments show that the reaction with Trp proceeds without isotopic scrambling of the label in the starting material [1-O-18]DMAPP. However, in the case of 4-fluorotryptophan, significant isotopic scrambling is observed (nu(PIX)/nu(rxn) = 1.1). This is consistent with a mechanism involving a discrete carbocation intermediate. Finally, a significant solvent kinetic isotope effect of 2.3 was observed in D2O, indicating that a proton transfer step is rate-limiting. Taken together, these observations support a mechanism that is a hybrid of mechanisms A and C. Ionization of DMAPP generates a dimethylallyl carbocation, and deprotonation of the indole nitrogen accompanies or precedes the nucleophilic attack.
引用
收藏
页码:7733 / 7739
页数:7
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