Study On Molecular Evolution Mechanism Of 3-Hydroxykynurenine Transaminase

3-hydroxykynurine transaminase(HKT)is a species-specific enzyme of diptera mosquitoes,and involved in the catabolism of 3-hydroxykynurine(3-HK),a toxic metabolic intermediate in mosquito tryptophan metabolism pathway.HKT is also an isoenzyme of alanine-glyoxalate aminotransferase/transaminase(AGT),which participates in the metabolism of glyoxylate.Xanthurenic acid(XA),the product of HKT enzyme reaction,is required for Plasmodium spp.development in the mosquito vectors.Therefore,an inhibitor of HKT may not only be a mosquitocide but also a malaria-transmission blocker.As a mosquito species-specific enzyme and plays an important role in mosquito detoxification metabolism,HKT must have experienced a unique evolutionary process.However,the molecular evolution mechanism of HKT is still unclear.In order to reveal the evolutionary origin and route of HKT,the molecular evolution mechanism was investigated for the first time in this study.By identifying HKT homologous in protozoa and metazoan and building phylogenetic tree,we first found that HKT was a paralog of AGT within mosquitoes.The topology of phylogenetic tree and synteny analysis indicated HKT gene was originated from an independent gene duplication event in the early stage of mosquito evolution.The parent gene was the common ancestor of mosquito HKT and AGT genes,and they experienced functional divergence after duplication.By analyzing the gene structures of HKT and AGT of mosquito species,it was found that HKT and AGT genes experienced different intron gain/loss events,resulting in changes in the coding region and non-coding region,which may lead to functional divergence.Functional divergence analysis showed that there was significant type I functional divergence between HKT and AGT.Due to the unique function of HKT,the study assumed that HKT gene had experienced positive selection pressure.The selection pressure analysis showed that HKT gene was under strong positive selection pressure during evolution,and 41 significant positive selection sites identified were found to concentrated on the region around and within the critical active sites of HKT,indicating that these sites affect conformational stability in three-dimensional structure,as well as substrate binding and catalytic process,of which were crucial to the functional evolution of HKT.In order to get a better understanding of the evolution of HKT,the sequence of the last common ancestor enzyme(Ancestral mosquito AGT,Anc Mosq AGT)of HKT and AGT was deduced,and a three-dimensional homology model was established,Anc Mosq AGT was expressed using a prokaryotic expression system and the recombinant Anc Mosq AGT was obtained.Molecular docking and enzyme activity analysis showed the ancestral enzyme had the function of recognizing new ligands related to toxic metabolites before gene duplication.This study reveals the molecular evolution scenario of the mosquito-specific gene HKT and provides evidence for the dramatic biochemical evolution of the key detoxifying enzyme for the first time,also reveals potential sites that could target for hindering the detoxification function,which may be used in mosquito larvicide design.