Structural Insight Into The Catalytic Mechanism And Inhibitor Screening Of Juvenile Hormone Acid Methyltransferase From Silkworm,Bombyx Mori

Juvenile hormones and ecdysterone are two important regulatory hormones during insect growth and development.Ecdysterone is responsible for regulating insect metamorphosis,while juvenile hormones are responsible for maintaining insect larval state to avoid premature maturation and promoting insect reproductive system development.As a key enzyme in the synthesis of Juvenile hormone,JHAMT(Juvenile hormone acid methyltransferase)is an important research target for regulating the biosynthesis of Juvenile hormone in insects.Relevant studies have shown that JHAMT dysfunction in insects has a significant impact on the synthesis level of Juvenile Hormone(JH)in insects.At the individual level,the growth and development of insects are retarded,fecundity is weakened,and the quantity and quality of offspring insects are severely damaged.However,most of the current studies on insect JHAMT focus on its expression characteristics and physiological functions,while the research on its catalytic mechanism is still lacking.The development of crystallography provides a new platform for the study of the catalytic mechanism of protease.The catalytic mechanism of protease can be explained at the atomic level through the analysis of the protease structure and the annotation of the catalytic site.In addition,the structural analysis of protease macromolecules also provides a basis for the design and development of specific inhibitory and enhancing small molecules.In this study,Bombyx mori was used as a model to reveal the mechanism of JHAMT,a key catalytic enzyme in insect juvenile hormone synthesis,and to screen specific small molecule inhibitors of JHAMT.A class of insect JHAMT-specific small molecular lead compounds were designed and reconstructed by the method of protein structure combined with computer-assisted screening.These small molecular compounds can change the catalytic activity of JHAMT in insects and regulate the synthesis level of juvenile hormones in insects,thus further regulating the growth and development of insects.The research results are as follows:1.Structural analysis of juvenile hormone acid methyltransferase JHAMT binding substrate complexThe co-incubated JHAMT-SAH-MF complex crystal was obtained by gas phase diffusion method,and the crystal was composed of six subunits.the cofactor SAH(S-(5’-adenosyl)-l-homocysteine is located horizontally between the two subunits of JHAMT,while the substrate small molecule MF((E,E)-famesoate)is located in the funnels of hydrophobic pockets above the cofactor.Superposition analysis of crystal topological structure shows that: JHAMT induces the cofactor SAH to enter the substrate binding pocket,which leads to the rearrangement of surrounding hydrophobic amino acids,and then forms a fungal-shaped hydrophobic pocket for the substrate to enter.After the substrate enters,the hydrophobic amino acids rearrange again to tighten the substrate binding pocket,promoting the enzymatic catalytic process of JHAMT and avoiding the solvation of substrate small molecules.2.Analysis and mechanism of JHAMT action siteThe enzyme activity parameters of amino acid residues involved in JHAMT catalytic process were determined by site-directed mutagenesis of amino acids combined with ultra-high performance liquid chromatography.By comparing the values of Km,Kcat and Kcat/Km in the catalytic process of protease,the differences of enzyme speed and catalytic efficiency between different mutant proteins and wild-type proteins were illustrated.The results showed that the enzyme catalytic efficiency of the mutant protein decreased to varying degrees,while the mutant protease of the amino acid residues Gln14/His114 had no methylase activity.The T15 Q reverse mutation of SILKWORM JHAMT3 showed that Gln14 mutation was one of the reasons for the loss of methyl transferase activity of homologous proteins JHAMT3,JHAMT4,JHAMT5 and JHAMT6.The binding ability of JHAMT to the substrate small molecule FA((E,E)-Famesoic acid)revealed the role of Gln14/His114 amino acid residues in the catalytic process of JHAMT.At the atomic level,the catalytic mechanism of JHAMT in insects is explained as follows: the substrate FA is located in a hydrophobic cavity,and the hydrophilic ester head is oriented towards the residues of Gln14/His114.The amino group on His114 and the amino group and hydroxyl group on Gln14 are bonded by hydrogen bond respectively,thus anchoring the substrate FA to a certain position.His114 then competes to take away the H atom on the hydroxyl group of substrate FA,leaving it in a state of degradation,which then attacks the nearby methyl donor SAM(S-(5’-adenosyl)-L-methionine)with a nucleophilic attack.The methyl group is taken away,and then the substrate is esterified to further produce MF.At the same time,MF can also be used as a synthetic precursor of JH,which can be further epoxidized into JH.3.Screening of small molecule specific inhibitors of insect JHAMTBased on the crystal structure of JHAMT,Schr(?)dinger software was used to screen the inhibitors of small molecules.The Pharmacophore model based on protein-ligand complex was constructed by using software,and pharmacophore was set by combining with the resolved catalytic action sites.The research group screened more than 130 small molecules with high matching degree from more than 500 small molecules for three times,and tested the inhibition efficiency of JHAMT for these small molecules.Using Bombyx mori,Spodoptera litura Fabricius,Aedes aegypti and Drosophila as research objects,ultra-performance liquid chromatography(UHPLC)was used to detect the inhibitory efficiency of small molecular compounds on the above four kinds of exogenically expressed insect JHAMT,and high inhibitory efficiency small molecules were obtained.In subsequent individual experiments,small molecules with lethal and disabling effects on Drosophila and Aedes aegypti were further screened out,but no significant inhibition was observed in Bombyx mori and Spodoptera litura Fabricius individual experiments.In vitro culture of the lateral pharyngeal body of Bombyx mori showed that the small molecule of the added inhibitor could reduce the synthesis level of juvenile hormone by inhibiting JHAMT in insects.It is speculated that the differences in insect body size,lifestyle,feeding environment and life cycle length may be the reason why there is no obvious characterization of individual inhibitor test between Bombyx mori and Spodoptera litura Fabricius.