Machenistic Study Of N-9 Methyltransferase In KuCha

Caffeine belongs to xanthine alkaloids,which was found in many plants,such as tea and coffee.As a central nerve stimulant,low-dose caffeine could help people to get rid of sleepiness and stay awake,making it one of the most commonly used psychotropic drugs around the world.However,consumption of caffeine may lead to several side effects,including rapid heartbeat,muscle tremor,sweating,insomnia,anxiety and gastrointestinal disorders.Recent studies have discovered that theacrine,ametabolite of caffeine,shows little stimulatory effecton central nervous system.On the contrary,theacrine has diverse beneficial biological activities,including antidepression,memory and learning improvement,and prevention of nonalcoholic fatty liver disease.The biosynthetic pathway of caffeine in tea and coffee has been well studied: xanthine nucleosideis methylated at N-7 position,followed by removal of 5-aribose to form 7-methylxanthine;7-methylxanthine is further transformed into theobromineby methylation at N-3 position;in the end,a third methyl group is added on the N-1 position to yield caffeine.Compared to caffeine,theacrine has a carbonyl group at the 8-position and a methyl group at the N-position at the 9-position.But how caffeine generates theacrine remains unclear.Our collaborator He Rongrong’s team cloned three N-methyltransferases from a rare bitter tea(Kucha): Ck CS,Ck Tb S,and Ck Tc S.In vitro enzyme activity assay confirmed that Ck Tc S has peculiar N9-methyltransferase activity,In vitro enzyme activity test confirmed that Ck Tc S has peculiar N9-methyltransferase activity,which can methylate the N9 position of 1,3,7-trimethyl uricacid to theacrine.Sequencially,Ck Tc S is highly conserved with Ck CS and Ck Tb S,but its catalytic activity is significantly different.In order to elucidate the molecular mechanism of Ck Tc S,we conducted crystal structure study on Ck Tc S and Ck Tb S.Through high-throughput screening,we successfully obtained the crystals of Ck Tc S-SAH-1,3,7-trimethyluric acid ternarycomplex and Ck Tb S in the apo state,and determined the crystal structure.Compared with the structure of N-methyltransferase from coffee,the structures of tea Nmethyltransferase share a similar fold: both exist as homodimer with each protomer consists of a flexible N-terminal Capping domain and the C-terminal cataltic domain.The 1,3,7-trimethyluric acid is enveloped in the active site through both hydrogen bonding and van der Waals interactions,with N-9 approximate to the thioether moiety of SAH.Residues surrounding the binding pocket include Met-15,Tyr-24 from the cap domain and Phe-30,Thr-31,Tyr-157,His-160,Trp-161,Arg-226,Ile-241,Trp-242,Cys-270,Ile-318 and Phe-322 from the methyltransferase domain.Among these residues,Tyr-24,Thr-31,Arg-226,Trp-161 and His-160 form direct hydrogen bonding interaction with 1,3,7-trimethyluric acid,while Tyr-157,and Phe-322 sandwich the 1,3,7-trimethyluric acid moleculethrough a π-π-stacking interaction.Notablely,Thr-31 forms a hydrogen bond with the carbonyl at the C-8 position of 1,3,7-trimethyluric acid,which may stabilize the O-8 carbonyl group in an iminol tautomeric state,and facilitate attack of SAM for N9-methylation In addition.To investigate the substrate specificity of Ck Tc S,we superimposed the structure of Ck Tc SSAH-1,3,7-trimethyluric acid and Ck Tb S.Although the two are in different states,the residues involved in substrate recognition almost share the same conformation,indicating that the substrate binding does not cause large conformational changes in the pocket.Combined with the sequence alignment of Ck Tc S,Ck Tb S and Ck CS,we identified the key residues that affect the substrate specificity of Ck Tc S: Arg-226,Ile-241 and Cys-270.The in vitro enzymaticactivity test showed that single mutation of the corresponding residues on Ck Tb S lead to a moderate increase in reaction products,while the double and triple mutations further increased the N-methyltransferase activity to almost half of that of Ck Tc S.Finally,it was found that the caffeine N-methyltransferases from tea and coffee adopt a distinct substrate binding strategy even though they possess the same methylation activity,which provides further evidence for the convergent evolution of the N-methyltransferases from tea and coffee in terms of their enzyme structure.To sum up,in this project,we studied the structures of N-methyltransferases in tea,and explained the substrate recognition mechanism of a novel N9-methyltransferase,which not only deepened our understanding of N-methyltransferase family,but also pointed out a new direction for the development of low-caffeinated beverages.