Structure And Enzymatic Mechanism Of The Juvenile Hormone Methyltransferase BmJHAMTs In Silkworm,Bombyx Mori

Insects are the most diverse animals in nature,which is because of their various ways of survival and metamorphosis.In the long-term evolution,insects go through several ecdysis processes,larvae to pupa and pupa to adult periods.In general,their shapes,living habits,physiological characteristics as well as the internal organs changed dramatically during the different stages,and these changes are strictly controlled by the juvenile hormone(JH)and molting hormone(20E).JH is responsible for maintaining insect growth and 20 E plays important roles in controlling larval molting and the process of matemorphosis from larvae to pupa.Until now,the researchers pay more attention to the downstream transcriptional regulatory,but few data were reported regarding the structural and enzymatic mechanisms of the key enzymes involved in the hormone synthesis and metabolic pathways.Silkworm,Bombyx mori is an importantly economical insect,which is also the research model of the lepidopterous insects.Thus,it seems to be crucial to the study the key enzymes that involved in the hormone synthesis pathways,which can not only help us to regulate the live cycles of the silkworms and further improve their main economic characters,but also provide important targets for the biological control of pests and new pesticide development.In this study,we focused on the key rate-limiting enzyme,Juvenile hormone acid methyltransferase(JHAMT),in the JH synthesis pathway of silkworm.On the one hand,we propose a systematic identification about the composition and distribution of JHAMTs family in B.mori.On the other hand,we determined the three-dimensional structure of BmJHAMTs,which has the vital significance for understanding the catalytic mechanism at the atomic level.In summary,this work would provide a structural basis for the development of friendly biological insecticides,especially for lepidoptera pests.The main results are as follows: 1.Bioinformatics analysis and expression characteristics of BmJHAMTs in silkwormAt first,we obtained the seven JHAMT gene in the B.mori genome(the JHAMT and six JHAMT-like genes,JHAMT-L1 to JHAMT-L6)by the bioinformatics analysis.The phylogenetic tree reveals that the different copies of the BmJHAMTs gene and lepidoptera insects grouped well together,what's more the BmJHAMT shared the higher similarity with that of Samia cynthia ricini,and the other six JHAMT-like genes grouped together into an additional branch in an irregular manner,which showed that the BmJHAMT-Like genes were little conserved in the B.mori.The sequence alignment revealed the BmJHAMTs shared 40-50% amino acid identity and had a conserved S-adenosyl-L-methionine(SAM)binding domain.The expression pattern of BmJHAMTs genes were identified using quantitative real time-polymerase chain reaction(RT-qPCR).The stage-specific expression analysis showed that BmJHAMT and BmJHAMT_L1 have the similar profile,both of them expressed in a low level at the transcript levels during the larval stage,and then increased at the wandering period.In addition,the two genes were expressed higher at each beginning of instar larval and then declined rapidly before the ecdysis period,corresponding to the Juvenile Hormone titers in hemolymph of the slikworm.BmJHAMT_L2 and BmJHAMT_L3,were mainly highly expressed at the larval stage.The expression level of BmJHAMT_L4 was undetectable from the first larval to the third larval stage,and then sharply increased in the 4th and 5th larval stage,which is also consistent with the JH titer.The BmJHAMT_L6 was predominantly detected in the larvae stage and was weakly identified from pupa to adult period.From the tissue-specific expression,the result showed that the BmJHAMT was mainly expressed in the Corpora allata(CA)either in the second day of 4th larval stage or in the fourth day of 5th larval stage.Whereas,the expression of BmJHAMT_L1 were rather ubiquitous in every tissues.We also found that the BmJHAMT_L2 and BmJHAMT_L3 primarily expressed in the silk gland(Sg).On the contrary,an obvious amount of BmJHAMT_L4 and BmJHAMT_L6 mRNA was detected in the fat body(Fb),epidermis,midgut(Mg)and so on.These findings suggest the peripheral tissues also exist the BmJHAMTs mRNA,which is inconsistent with the long-accepted paradigm that BmJHAMT was only expressed in the CA.These BmJHAMTs proably exert the methyltransferase activity in peripheral tissues and catalyze JH acid to JH.2.Prokaryotic expression,purification and Enzymatic properties of BmJHAMTsWe cloned the ORF of BmJHAMTs(BmJHAMT,BmJHAMT_L1 to L4 and BmJHAMT_L6)from the mix cDNA of CA,Fb and Sg at the second day of 4th instar larval stage,and then ligated them into the prokaryotic expression vector.The recombinant plasmids contained each Bm JHAMTs gene were transformed into E.coli strain and the BmJHAMTs proteins were successfully expressed at 16 ?.The recombinant BmJHAMTs proteins were purified by Ni-NTA column affinity chromatography and gel filtration chromatography.The purified BmJHAMTs proteins were stored,which could be used for crystallization and enzymatic assays.The methyltransferase activity of recombinant BmJHAMTs proteins was performed by HPLC.The commercial substrate farnesoic acid(FA,a similar structure of JH acid)restitute of JH acid was added and incubated with BmJHAMTs in the presence of SAM.The results indicated that the BmJHAMT and BmJHAMTL1 BmJHAMT showed activity towards FA,and the has the BmJHAMT had a higher activity than that of BmJHAMT_L1.In contrast,no enzymatic activity was detected for the rest of BmJHAMTs(BmJHAMT_L2 to L4 and BmJHAMT_L6).We proposed several reasons for these BmJHAMTs : Firstly,there was no post-transcriptional and post-translational modification of these proteins in the prokaryotic expression system,which showed no catalytic activity in vitro.Secondly,it was possible that these proteins are kind of precursor enzyme and should be activated in the body of the B.mori.Lastly,we speculated that these proteins accommodate and catalyze different substrate rather than the juvenile hormone acid.3.The structure of BmJHAMT insight into its enzymatic mechanismAs we all know,the late-step enzym in the JH synthesis pathway were highly specific to insects,but non three-dimentional structure of JHAMT among the insects were resolved.In order to obtain the first structure of JHAMT,we screened their crystals by the kits from Hampton Research.Through the optimization of BmJHAMTs crystallization and the X-ray diffraction,we finally determined the crystal structures of the apo-BmJHAMT_L1,BmJHAMT_L1-SAM complex and the apo-BmJHAMT_L2.The structure of BmJHAMT-L1 was consist of a conserved ?/? Rossmann domain.This Rossmann fold domain contains a seven-stranded ? even-strand sandwiched between two helical regions and it is a typical fold for SAM-binding domain in methyltransferase.On the top of the SAM-domain,the g-helix regions form a ?lid-like? shape,which could be related to its specific substrate binding(FA or JHA).Comparison of the structure of apo-BmJHAMT_L1 and BmJHAMT_L1-SAM complex revealed that the binding of substrate SAM would make the significantly conformational change.The loop(from Asp111 to 119)was swung and tended downward,playing an important role for the insert of SAM and substrate binding or specificity.To characterize whether SAM was binding within the active site pocket through hydrogen bond networks,we mutated these residues and measured their binding affinity(Kd)by Fluorescence spectrophotometry assay.The result showed that the mutation of Asp68 and Thr113 resulted in an obvious decrease in affinity efficiency,suggesting these sites were crucial for stabilization of SAM.To identify the binding site of substrate JHA,we docked the JHA molecule in to the structure of BmJHAMT-SAM.In the docking mode,the carboxyl groups of JHA faced to the outside of the substrate-binding pocket and fitted nicely to form hydrogen bonds with the Gln15,His115 and Trp116.Whereas the carbon chains of JHA situated in the bottom of the hydrophobic pocket,and stabilized by Val147,Phe148,Asp205,Val207,Leu216 and Leu256 vis hydrophobic interaction.Subsequently,we would mutate these residues and identify their catalytic activity to further prove this hypothesis and determine the enzymatic mechanism of BmJHAMTs.