Crystal Structure Of MarI,A C-methyltransferase Involved In ?-methyltryptophan Biosynthesis

Methyltransferases is one of the most diverse in nature and important modification enzymes.They can utilize SAM as a methyl donor to catalyze the methylation of a series of compounds ranging from small metabolites to macromolecules.?-Methyl tryptophan??-Me Trp?is the important building block and biosynthetic precursor of natural products which show great bioactivities of antiviral,antibacterial and antineoplastic properties.Compared to the other ?-methyl amino acids,?-Me Trp has more modification sites to enhance the biological activities of the ?-Me Trp-containing compounds due to the unique indole ring structure.Our previous work has shown that the methylation of ?-carbon catalyzed by Mar I or its homologous protein StnQ1 is the key step of ?-Me Trp biosynthetic pathway.Mar I or StnQ1 utilizing SAM as the donor,catalyzes the methylation reaction of the Indole pyruvic?Inpy?,which generate SAH and?3R?-?-methyl indole pyruvate??-Me Inpy?that is converted to?2S,3R?-?-Me Trp in the catalysis of the aminotransferase.To elucidate the reaction mechanism of Mar I,we carried out the crystal structural study of this enzyme.We obtained two ternary complex structures of Mar I-SAM-Inpy and Mar I-SAH-?-Me Inpy which both diffracted to 2.3 ?.Mar I was also confirm to be a Fe³⁺-dependent C-methyltransferase,and the ferric ion was hexacoordinated.In the complex structure of Mar I-SAM-Inpy,the ?-amino group?NH₂?of the methionine moiety of SAM was coordinated with the ferric ion,which is unique for methyltransferases.The related biochemical characterization showed that this kind of coordination can improve the stability of the ferric ion in protein.However,the distance between the methyl group of SAM and the ?-carbon of the methyl acceptor was 7.9 ?,which is unreasonable for methylation.In the complex structure of Mar I-SAH-?-Me Inpy,an additional water replaced the role of the ?-amino group?NH₂?of SAM methionine moiety and coordinated to the ferric ion and SAH wasn't coordinated with the ferric ion.The distance between the methyl group of SAM and the ?-carbon reduced to 5.3 ?.Combining the two structure with QM/MM,we speculated that the coordination bond between SAM and Fe³⁺ was broken during the reaction process,then conformation of SAM methionine was changed and pulled away the center of active-site pocket,which lead to the shorter reaction distance to facilitate the methylation.Furthermore,Trp118 and Dsp262 were determined to be the key residues to facilitate the methylation.