3,4-Dihydroxyphenylacetaldehyde synthase evolved an ordered and distinct active site to promote elastic cuticle formation and blood intake in Aedes aegypti

doi:10.21203/rs.3.rs-2667972/v1

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3,4-Dihydroxyphenylacetaldehyde synthase evolved an ordered and distinct active site to promote elastic cuticle formation and blood intake in Aedes aegypti

https://doi.org/10.21203/rs.3.rs-2667972/v1

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3,4-Dihydroxyphenylacetaldehyde synthase (DHPAAS) catalyzes the direct conversion of 3,4-dihydroxyphenylalanine to 3,4-dihydroxyphenylacetaldehyde (DHPAA), an important intermediate in the formation of flexible insect cuticle. In order to clarify the precise roles DHPAAS plays in insect development and survival, DHPAAS was characterized throughout the physiological to the molecular levels. Extensive in vivo experiments in Aedes aegypti confirm that DHPAAS is essential for blood feeding, egg development and cuticle structure formation. The crystal structure of insect DHPAAS was then solved to reveal the structural basis underlying the catalytic production of the key cuticle intermediate DHPAA. The molecular view shows a DHPAAS active site that is distinct from that of the homologous enzyme 3,4-dihydroxyphenylalanine decarboxylase. Stabilization of the flexible 320–350 region is observed to position the 350–360 loop towards the catalytic asparagine residue, and these distinct features are suggested to promote pyridoxal 5'-phosphate-dependent amine oxidation. Additional molecular dynamics simulations further support the involvement of Phe82, Tyr83 and Asn195 in substrate binding and catalysis, and also shows increased fluctuations limited to loop residues 330–345 in Aedes aegypti DHPAAS.

Biological sciences/Structural biology/X-ray crystallography

Biological sciences/Biochemistry/Enzymes/Oxidoreductases

Biological sciences/Zoology/Entomology

α-methyldopa-resistant protein

4-dihydroxyphenylacetaldehyde synthase

Aedes aegypti

flexible cuticle

blood feeding

mosquito

Insect L-3,4-dihydroxyphenylalanine decarboxylase (DDC) is a pyridoxal 5’-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AAAD) that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (dopa), 5-hydroxyl-trptophan and structurally-similar aromatic amino acids ¹. While mammals often possess a single copy of AAAD, insect genomes contain multiple copies of AAAD and related genes. Drosophila melanogaster contains five AAAD-related genes, including one typical DDC, two tyrosine decarboxylase related sequences, and two sequences annotated as α-methyldopa (AMD)-resistant (NP_476592 isoform A and NP_724162 isoform B). Accordingly, AAAD function in insects is diverse with roles in neurotransmitter biosynthesis ^{2, 3}, immune responses ^{4, 5}, cuticle sclerotization ^{6, 7} and egg-tanning ⁸.

Previous studies suggested that two Aedes aegypti homologues of D. melanogaster AMD-resistant protein, encoded by AAEL010734 and AAEL010735, play an essential role in the assembly of the mosquito cuticle ^{9, 10}. Although the two Ae. aegypti AMD-resistant proteins share high sequence identity (51%) to Ae. aegypti DDC (AeDDC) (XP_001648264.1), both of these enzymes were found to convert dopa to 3,4-dihydroxyphenylacetaldehyde (DHPAA, or dopal), rather than producing dopamine. Therefore, the Ae. aegypti AMD-resistant protein has been named DHPAA synthase (DHPAAS, or dopal synthase). The transcriptional profile of Ae. aegypti DHPAAS (AeDHPAAS) closely correlates with cuticle development, and the catalytic product of AeDHPAAS is the reactive aryl acetaldehyde DHPAA that can crosslink proteins and other components of the insect cuticle ^{9, 11}. Recent studies demonstrated that AeDHPAAS affects larval development and adult survival in Ae. aegypti via RNA interference (RNAi)-mediated gene knockdown ¹².

The specialized function of D. melanogaster AMD-resistant proteins remained a mystery until the mosquito homologues were characterized with DHPAAS activity ⁹. Early studies reported that the gene encoding AMD-resistant protein in D. melanogaster, confers resistance to the toxic DDC inhibitor AMD ^{1, 13}. Furthermore, a mutation in the gene encoding AMD-resistant protein was also reported to cause structural defects in the cuticle ^{11, 14, 15}. Subsequent studies finally showed that both D. melanogaster AMD-resistant proteins exhibit DHPAAS activity ^{9, 16}.

There seems to be a consensus that the DHPAAS enzymatic product DHPAA is involved in the assembly of un-melanized, flexible cuticle, however the precise role of DHPAAS in cuticle formation and the catalytic mechanism of DHPAAS have not been clarified. Furthermore, although the reaction which DHPAAS catalyzes is distinct from that of the homologous enzyme DDC ⁹, this specialized enzyme continues to be misannotated as DDC or AAAD in most public databases and even in some literature. While the crystal structure of D. melanogaster DDC (DmDDC) has been reported ¹⁷, there are no reported crystal structures of DHPAAS.

The current study aims to clarify the precise function of DHPAAS in insect cuticle formation, by characterizing DHPAAS throughout the physiological level down to the molecular level. Physiological investigations confirm that AeDHPAAS is essential for the development of flexible insect cuticle, where AeDHPAAS knockdown impairs blood intake, abdominal elasticity, egg hatching and cuticle structure at the µm level. To understand the molecular level, the crystal structure of D. melanogaster DHPAAS (DmDHPAAS) in complex with PLP was solved, revealing a more ordered 320–350 region in comparison to that of homologous DmDDC. The relatively ordered 320–350 region appears to influence movement of the Leu353- and Phe103-containing loops towards and away, respectively, from the catalytic Asn192 residue of the neighboring DmDHPAAS subunit. Molecular dynamics using the full length AeDHPAAS model further supports the involvement of Tyr83, Phe82 and Asn195 in substrate binding and catalysis, and shows increased fluctuations limited to loop residues 330–345. These distinct structural features of insect DHPAAS provide a structural basis underlying the catalytic production of the essential cuticle intermediate DHPAA.

Abdominal elasticity varies between male and female adult mosquitoes

Before selecting adult mosquitoes for AeDHPAAS knockdown experiments, male and female adult mosquitoes were first compared for their ability to handle abdominal injections. The abdomens of both male and female adult mosquitoes were flat before the injection of Ringer’s solution (Fig. 1a, d). After injection with Ringer’s solution, the abdomens of both male and female adults swelled (Fig. 1b, c, e, f). These initial results showed that female adults could survive after injection of 3–5 µL solution, but male adults could only survive after being injected with 0.3 ± 0.05 µL solution at most. Figure 1b shows a surviving adult male after injection with 0.23 ± 0.05 µL of Ringer’s solution. In contrast, Figs. 1e and 1f show surviving adult females after injection with 3 µL and 4 µL of solution, respectively. Based on the abdominal injection results, female adult mosquitoes were selected for subsequent RNAi experiments.

DHPAAS knockdown increases mortality and reduces blood meal volume

Adult female mosquitoes were collected 12 h after RNAi microinjection to analyze gene expression. Real-time PCR (qPCR) showed no significant difference between negative and blank controls, while the relative expression of AeDHPAAS in the RNAi group was significantly lower (75% lower, P < 0.001) than that in the control groups (Fig. 2a). Meanwhile, almost no adults in the control groups died after 4 µL of Ringer’s solution injection, but 50% (P < 0.0001) of the adults with AeDHPAAS knockdown failed to survive (Fig. 2b). The weight of adult mosquitoes before and after blood meal was measured, as shown in Fig. 2c, and no significant weight differences were observed between control and experimental groups. The average weight of each female adult was 1.7–1.8 mg (Fig. 2c). Figure 2d illustrates blood meal intake weight, the blood meal weight of adults in the control groups ranged from 3.7 mg to 4 mg, while the blood meal weight of adults with AeDHPAAS knockdown was only 2 mg (P < 0.0001).

DHPAAS knockdown disrupts the abdominal cuticle

After RNAi knockdown of AeDHPAAS, followed by oviposition, 40 ± 3.3% of the adult female mosquitoes died within three days (Fig. S1). In contrast, only 5% failed to survive in the control groups under matching conditions. Furthermore, 25 ± 5% of the surviving mosquitoes with knocked-down AeDHPAAS showed lowered tolerance to the abdominal injection of Ringer’s solution, with a higher frequency abdominal rupturing in mosquitoes with knocked-down AeDHPAAS (Fig. 3).

DHPAAS knockdown reduces egg hatching

RNAi knockdown of AeDHPAAS had no significant effect on the number of eggs, where each mosquito produced 100 ± 20 eggs (Fig. 4a). However, hatching of eggs was significantly inhibited by AeDHPAAS knockdown. The egg hatching rates of the control groups and AeDHPAAS‑dsRNA treated group were 90 ± 5% and 53 ± 28%, respectively (Fig. 4b). Approximately 60% of the non-hatching eggs of the AeDHPAASdsRNA treated group contained no larvae, but all eggs of the control groups contained developed larvae. These non-hatching eggs without larvae in the AeDHPAASdsRNA treated group rapidly dehydrated after reducing humidity from 20–10%; control group eggs did not dehydrate with decreased humidity (Fig. 4c). The remaining (~ 40%) non-hatching eggs of the AeDHPAASdsRNA treated group contained formed larvae and did not dehydrate; however, none of these eggs could hatch.

DHPAAS knockdown alters cuticular structure

During mosquito cuticle formation, the epithelium first secretes cuticulin and then the lamellate endocuticle. Finally, the exocuticle is derived by quinone tanning of the outer lamellae of the endocuticle ¹⁸. To observe the effect of DHPAAS knockdown on cuticle formation, transmission electron microscopy (TEM) was used to observe abdominal cuticular structures 12h after microinjection. The resulting TEM images showed that the endocuticle was thicker than the exocuticle in the control groups (Fig. 5a, b). In contrast, the AeDHPAAS RNAi group showed a thinner endocuticle layer relative to the exocuticle (Fig. 5c).

Differential gene expression after DHPAAS knockdown

Knockdown of AeDHPAAS resulted in obvious abnormal exfoliation and delayed development of Ae. aegypti larvae. To further explore the functional role of AeDHPAAS in larval development and molting, gene expressions of AeDHPAAS RNAi and control groups were focally analyzed using RNA-seq. Genes that were significantly differentially expressed between the experimental group and both negative and blank control groups could then be analyzed. After AeDHPAAS knockdown, the development of larvae was significantly inhibited ¹². The differentially expressed genes involved in development were analyzed according to predefined pathways annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). As a result, 60 development-related genes were found to be differentially expressed after AeDHPAAS knockdown, of which only five genes were up-regulated while the rest were down-regulated (Fig. 6a).

Our previous research has shown that knockdown of AeDHPAAS can cause abnormal larval molting ¹². Accordingly, the expression of genes related to cuticle proteins, chitin metabolism and lipid metabolism were altered after knockdown of AeDHPAAS. Compared with the control groups, 118, 107 and 110 genes related to cuticle proteins, chitin metabolism and lipid metabolism, respectively, were differentially expressed (Dataset S1) (Fig. 6b-d). Most of these differentially expressed genes showed trends of up-regulated expression. Nine out of 118 differentially expressed cuticle genes related to cuticle proteins were downregulated (Fig. 6b), 24 out of 107 genes involved in chitin metabolism were downregulated (Fig. 6c), and 27 out of 110 genes involved in lipid metabolism were downregulated (Fig. 6d). In addition, interesting changes in the expression of sterol carrier protein 2 (SCP-2) and lipophorin receptor (LpR) genes were observed in Ae. aegypti. Five out of eight SCP-2 genes were significantly upregulated and a single LpR gene was significantly downregulated (Fig. 6e).

Crystal structure of insect DHPAAS

To provide insight into the structure-function relationship and catalytic mechanism of DHPAAS, DmDHPAAS was purified and crystallized for X-ray crystallographic analysis. After extensive optimizations, the enzyme could finally be well-crystallized. The structure of DmDHPAAS was solved using molecular replacement based on the DmDDC structure (PDB ID 3K40) ¹⁷. The crystal structure was refined to 2.2 Å resolution with good crystallographic statistics (Table S1) and the structure was deposited and released (PDB ID 6JRL). According to analysis of the protein interfaces, surfaces and assemblies service ¹⁹, the DmDHPAAS crystal structure is a dimer (Fig. S2). The DHPAAS structural architecture resembles other type II PLP-containing enzyme structures. Similar to the DmDDC crystal structure, which contains a disordered region spanning residues 322–348, residues 328–341 of the DmDHPAAS crystal structure are disordered.

The DHPAAS active site is located at the monomer-monomer interface of the homodimer rebuilt based on the symmetry, and is mainly composed of residues from a single subunit; however, the Phe103- and Leu353-containing loops support the formation of the active site of the neighboring subunit. The active site Lys303 is observed to form a Schiff base with the PLP cofactor, and this internal aldimine is referred to as lysine-pyridoxal-5-phosphate (LLP) (Fig. 7).

Similar to most PLP-dependent enzymes, the LLP protonated pyridine nitrogen forms a salt bridge with the Asp271 carboxylate. Furthermore, the LLP pyridine ring is anchored by the Ala273 methyl group as well as the catalytic Asn192 amide group. The LLP pyridine ring hydroxy group is adjacent to the Thr246 side chain and two water molecules. The LLP phosphate moiety is further stabilized by multiple interactions with Ser149, Asn300 and His302 from within the same subunit, and is 5.0 Å from Leu353 of the neighboring subunit (Fig. 7a).

Structural comparison of DHPAAS and DDC

There is a significant difference regarding the loop containing the key active site residue Phe103, when comparing DmDDC and DmDHPAAS structures. As shown in Fig. 7b (right panel), Phe103 is relatively close to LLP and His192 in DmDDC; however, in DmDHPAAS the Phe103-containing loop is oriented away from the active center. His192 or Asn192 are important for determining DmDDC or DmDHPAAS catalytic activity, respectively ^{9, 20}; where His192 is much closer to Phe103 (4.2 Å) in the DmDDC complex compared to the corresponding Asn192 distance in DmDHPAAS.

The DmDHPAAS region spanning residues 320–350 displayed a more defined structure as compared to that of DmDDC (Fig. 7b, left panel). In the insect DmDDC structure, residues 322–348 (26 residues) are disordered and cannot be observed; however, in the DmDHPAAS structure, the disordered region only includes residues 328–341 (13 residues). The stabilized 328–341 loop is observed to influence the Leu353-containing loop adjacent to the DHPAAS active site; DmDHPAAS Leu353 residue is positioned just 3.4 Å away from the catalytic Asn192 residue, whereas the corresponding Leu350 residue in DmDDC is oriented much further away from the active site (Fig. 7b, left panel). The DmDHPAAS Leu353-containing loop also contains Gly354 which has been reported to be important for substrate specificity towards dopa ²¹.

AeDHPAAS full-length model

To construct a valid model, the crystal structure of DmDHPAAS (PDB ID 6JRL, chain A) was selected as the most satisfactory template, with sufficient homology to AeDHPAAS (61% identify and 92% coverage). The MODELLER program was used to build a homology model of AeDHPAAS. The plotted Discrete Optimized Protein Energy (DOPE) score profile shows regions of relatively high energy for the disordered region spanning from residues 330 to 345, and the long loop at the N-terminal (Fig. S3). This disordered region is located away from the active site with an approximately 40 Å distance between Gln341 and Asn195 throughout the model. PROCHECK showed that 91.4% of residues are located in most favored regions, 7.5% of residues are located in additional allowed regions, and 0.4% of residues are found in outlier regions (Fig. S4). Verify3D indicated that 80.8% of amino acids show an averaged 3D-1D score ≥ 0.2. The above statistics indicate a reliable structure model.

Molecular docking and simulation of AeDHPAAS substrate binding

To reveal possible ligand-enzyme interactions, detailed analysis of substrate binding to the AeDHPAAS active site was carried out with a molecular docking bound ligand. AutoDock Vina docked dopa, DHPAA and dopamine into the active site cavity (Fig. 8). The results show that dopa is a good ligand with the highest relative binding affinity among the three studied ligands (-5.6 kcal/mol) and the docking solutions of DHPAA and dopamine resulted in binding affinities of -5.0 kcal/mol and − 4.9 kcal/mol, respectively. The substrate-binding pocket of AeDHPAAS is composed of a set of conserved residues, as well as three variable residues (Phe82, Tyr83 and Asn195) that have diverged from the AeDDC sequence. The substrate specificity determining residues appear to be similar between DDC and DHPAAS. Phe82-Tyr83 is of particular interest as this motif is conserved as Phe-Tyr in AeDHPAAS, but substituted to Tyr-Phe in AeDDC. Asn195 is conserved in AeDHPAAS, but substituted to histidine in AeDDC. Substitution of these three variable residues is reported to tune bifunctional switching between DHPAAS and DDC activities ^{21, 22}.

Considering the dimeric conformations in a physiological environment, molecular dynamics was used to calculate the atomical dynamic movements and conformational variations of AeDHPAAS dimer bound to ligands and LLP cofactor (Fig. 9). Root-mean-square deviation (RMSD) plots (Fig. 9a-c) indicate convergence in all simulations. The fluctuations occurring during simulations may be due to loop regions with higher flexibilities. The local motility properties of amino acid residues in AeDHPAAS complexed with ligands were further determined by the root-mean-square (RMS) fluctuation. Chain A experiences less conformational changes than chain B according to the RMSD and RMS values (Fig. 9a-f). Overall, it is observed that there are two main areas with high-RMS fluctuations, including the N-terminus and residues 328–341 (Fig. 9d-f).

DHPAAS has been proposed to be involved in insect cuticle formation since the discovery that the encoding gene confers AMD-resistance ¹. In the current study, DHPAAS was characterized throughout the physiological to the molecular levels, confirming the direct involvement in cuticle formation. The essential role of DHPAAS in insect survival was also observed through influences on blood intake and egg hatching.

The physiological results revealed an abdominal phenotype in DHPAAS-knocked-down mosquitoes injected with Ringer’s solution (Fig. 3). This could indicate either a weakened abdominal cuticle, or a decrease in cuticle flexibility and elasticity, where both possibilities indicate defects in cuticular cross-linking. Furthermore, a weaker or less flexible cuticle was also observed in male mosquitoes (Fig. 1), and this is consistent with our previous finding that AeDHPAAS is expressed at lower levels in males ¹².

Insect cuticle is mainly composed of cuticle proteins, chitin and lipids ^{23, 24}. Previous studies have found that the silencing of genes encoding insect cuticle proteins can lead to developmental defects that prevent normal molting and metamorphosis ^{25, 26, 27}. The RNA-seq data of this current study shows that AeDHPAAS silencing altered the expression of 118 genes encoding cuticle proteins, 107 genes involved in chitin metabolism and 110 genes involved in lipid metabolism. Surface lipids play a fundamental role in insect growth and development and also significantly affect the structure and physical properties of insect cuticle ^{28, 29}. Moreover, the transport of lipids is coordinated by lipid transport proteins, and therefore, these proteins are also crucial in maintaining lipid balance and development. These observations suggest that AeDHPAAS plays direct or direct roles in regulating the expression of diverse genes that are responsible for producing major cuticle components in Ae. aegypti.

To understand the molecular basis for DHPAAS function, the first insect DHPAAS crystal structure was solved at a resolution of 2.2 Å in this study. The AeDHPAAS structure and catalytic mechanism could then be investigated with molecular modeling, docking and dynamics simulations. Comparison of the DHPAAS structure to the structure of monoamine oxidase (MAO), which catalyzes a similar reaction, supports additional roles for active site aromatic residues. MAOs A and B contain an aromatic “sandwich” of two tyrosine residues that are believed to be involved in the oxidation of the substrate amine ³⁰. The MAO related enzyme L-amino acid oxidase (LAAO) also contains a similar aromatic “sandwich” composed of two tryptophan residues ³⁰. Therefore, many aromatic amino acids observed around the DmDHPAAS active site, including Trp71, Tyr79, Tyr80, Phe103 and Phe309, might play important roles in DHPAAS catalytic activity.

DHPAAS homologues have also been shown to convert aromatic amino acids to aryl acetaldehydes in several model plants ³¹. While DHPAAS-like enzymes are predicted to be present in numerous other plant species, their functions remain speculative. Recently DHPAAS from Bombyx mori has been applied to the efficient biosynthesis of benzylisoquinoline alkaloids in Escherichia coli^{22, 32}, raising an intriguing speculation about the possible involvement of DHPAAS or related enzymes in plant alkaloid pathways.

The current study provides a comprehensive view of DHPAAS function throughout the physiological level down to the molecular level. The physiological studies confirm the role of DHPAAS in elastic cuticle formation, which is essential for blood intake and survival. The molecular view of the distinct DHPAAS crystal structure contributes towards a more complete understanding of PLP-dependent oxidation mechanisms.

Abdominal injection of adult mosquitoes

Ringer’s solution (150 mM NaCl, 3 mM CaCl₂, 3 mM KCl, 10 mM N-Tris-methyl-2-aminoethanesulfonic acid, and 25 mM sucrose, pH 6.9) was injected into abdomen through the thorax of adult mosquitoes. Male adults were injected with 0.23 µL or 0.28 µL of Ringer's solution ³³. Female adults were injected with 2 µL or 3 µL of Ringer's solution.

RNAi knockdown of DHPAAS in adult mosquitoes

Purified dsRNA (2–3 µL) was injected into the thorax of adult mosquitoes 3–5 days after emergence, to knockdown transcriptional abundance according to a previous report ¹². dsRNA was injected into male and female adults. Each female adult was injected with about 2–3 µL dsRNA. For negative and blank control groups, respectively, E. coli β-glucuronidase (gus)-dsRNA and diethylpyrocarbonate (DEPC)-treated water were injected. Then the relative expression of AeDHPAAS was detected by qPCR. Subsequently, 12 h after the injection, all female adults were injected with about 4 µL of Ringer’s solution, and mortalities were accounted for. The surviving female adults were evaluated for weight of blood meal, and mosquito weight before and after blood meal, with a total of 50 mosquitoes selected for each male and female group. Within three days of a blood meal, sugar water was given ad libitum. After three days of feeding on blood, the weight of blood (calculated by the weight of adult mosquitoes after the blood meal minus the weight before the blood meal), the number of oviposited eggs, and their survival and hatching rate, for each mosquito group, were determined. This experiment was repeated three times.

RNAseq and analysis

In order to determine genes that are differentially expressed in AeDHPAAS knockdown mosquitoes, relative to control mosquitoes, RNA-seq analysis was performed as described in a previous paper ³⁴. The total RNA of 4th instar larvae was extracted by tissue homogenization in trizol reagent (Invitrogen, Shanghai, China). cDNA was prepared and then sequenced in BGI, China. Transcript abundance was determined using RNA-seq by Expectation Maximization (RSEM) ³⁵, where expression level read counts were normalized using Fragments Per Kilobase of transcript per Million mapped reads (FPKM).

Transmission electron microscopy

Abdominal cuticles of adult mosquitoes were harvested after RNAi and before blood meal. The harvested cuticles were fixed in 2.5% glutaraldehyde at 4°C overnight. The samples were washed three times for 5 min with phosphate buffer. Specimens were further fixed in 1% osmium tetroxide buffer in phosphate buffer (0.1 M, pH 7.0) at 4°C for 1–3 h. After fixation, specimens were washed three times with phosphate buffer (0.1 M, pH 7.0) for 5 min. The samples were gradually dehydrated with 30, 50, 70, 90, 95 and 100% ethanol for 5 to 10 min at each step; the 100% ethanol step was repeated three times to ensure complete dehydration.

After dehydration, the samples were embedded in epoxy resin (Sigma-Aldrich, St. Louis, USA). The specimens were sliced with an ultramicrotome (Leica UC6; Leica, Vienna, Austria). The sections were stained with uranyl acetate (Syntechem, Changzhou, China) and lead citrate (Acros, Shanghai, China) and observed using an electron microscope (JEM1200EX; Jeol, Tokyo, Japan).

Preparation of recombinant DmDHPAAS

Cloning and expression of DmDHPAAS from D. melanogaster cDNA were conducted according to previous reports ^{9, 20}. A DmDHPAAS (NP_476592, isoform A) cDNA sequence was amplified from D. melanogaster using a forward primer with a NdeI restriction site (5'- AAAACATATGATGGATGCCAAGGAGTTTCGGGAAT-3') and a reverse primer (5'- AAAACTCGAGTCACTGAGATTTCTCGTGCGTTG-3') with an Xho I site. The amplified full-length NP_476592 sequence was cloned into an Impact™-CN plasmid (New England Biolabs). E. coli ER2566 competent cells were transformed with the recombinant plasmid and cultured at 37 ^oC. After induction of a 2 L culture with 0.3 mM isopropyl-1-thio-β-D-galactopyranoside, the cells were cultured at 15^oC for 24 h to produce target protein with a fused chitin-binding domain. To inactivate serine and cysteine proteases, 1 mM phenylmethylsulfonyl fluoride was included in the lysis buffer. Recombinant protein was extracted from cells and applied to a column packed with chitin beads. Target protein, bound to the chitin beads via the fused chitin binding domain, was subsequently cleaved by incubation under reducing conditions at 23^oC for 24 h. Cleaved protein was eluted from the column and concentrated in a Centricon YM-50 centrifuge concentrator (Millipore). The affinity purification resulted in the isolation of 80% pure DmDHPAAS. Increased protein purity was achieved after Source Q ion-exchange and gel-filtration chromatographies. Purified DHPAAS was concentrated to 8 mg ml^− 1in 50 mM phosphate buffer (pH 7.5) in a Centricon YM-50 concentrator (Millipore). Protein purity was analyzed by SDS-PAGE, and protein concentration was determined with a Bio-Red protein assay kit (Hercules, CA).

Protein crystallization and structure determination

Protein crystals were grown in 2 µL hanging-drops, containing 1 µL of protein sample (8 mg/mL) and 1 µL of reservoir solution, suspended above reservoirs with 500 µl buffer. Optimal crystallization buffer consisted of 0.16 M MgCl₂, 0.085 M NaCl, pH 6.5, 25.5% w/v PEG 8000 and 15% glycerol. Individual crystals were cryogenized in crystallization buffer supplemented with glycerol to a final concentration of 20% for cryo-protection. The crystal structure of DmDHPAAS was determined using molecular replacement based on the reported structure of insect DDC (PDB ID 3K40) ¹⁷. Molrep ³⁶ was used to calculate cross-rotation and translation of the model. Iterative cycles of crystallographic refinement were performed with Refmac 5.2 and model building was performed with Coot 0.7.1 ³⁷. PLP cofactor was added after the R factor dropped to approximately 0.25 at full resolution, based upon 2Fo–Fc and Fo–Fc electron density. Solvent was automatically added and refined using ARP/warp ³⁸ and Refmac 5.2.

Building a full-length AeDHPAAS model

The full-length sequence of AeDHPAAS (XM_001661007) was aligned with DmDHPAAS (PDB ID 6JRL) to build a homology model of AeDHPAAS with Modeller 10.2 ³⁹. Models were evaluated for their quality using various structural parameters and comparative evaluation tools, such as optimized protein energy (DOPE) and molecular PDF (molpdf), PROCHECK and Verify3D program from the SAVES server (https://saves.mbi.ucla.edu/). Low molpdf and DOPE assessment values were considered to indicate better quality models. PROCHECK was used to check the stereochemical quality of the resulting model. Verify3D was used to determine the compatibility of an atomic model (3D) with its own amino acid sequence (1D). The best model was selected and refined with energy minimization to optimize stereochemistry using the loop model script of MODELLER 10.2. The optimized model was then used for docking and molecular dynamics simulations.

Molecular docking and simulation

AutoDock Vina ⁴⁰ produced molecular docking solutions for dopa, DHPAA and dopamine as ligands of AeDHPAAS. Ligands and receptor were prepared with AutoDock Tools 1.5.6 (http://mgltools.scripps.edu/). The coordinates of LLP and the AeDHPAAS-LLP complex were obtained by superposing the DmDHPAAS crystal structure (PDB ID 6JRL) onto the newly built model. Partial charges for protein atoms were assigned using Kollman charges, while LLP and ligands were assigned with Gasteiger charges, making sure that presented integer charges were included for all atoms. A grid box (34 × 20 × 24 Å) with 1.0 Å spacing covered the entire active-site cavity around LLP.

PLIP ⁴¹ and Pymol (https://pymol.org/2/) were used to visualize the docking results. Protein-ligand complexes resulting from docking were validated with molecular dynamics using Gromacs (version 2021). Topology and parameter files for AeDHPAAS were generated using the CHARMM36 force filed ⁴², while topology and parameter files for ligands were generated by the CHARMM General Force Field (CGenFF) program ⁴³. TIP3P water molecules were included and with neutralization by Na⁺ ions. Dodecahedron periodic water boxes were simulated with a margin of 1.0 nm. The solvated structure was minimized by the steepest descent algorithm until the maximum force reached < 1,000 kJ/mol/nm at 300 K. Equilibration of the complex was performed for 100 ps with 300 K using V-rescale (modified Berendsen thermostat) as NVT ensemble, the system was then equilibrated at 1 atm pressure using Berendsen algorithm NPT ensemble for 100 ps. The LINCS algorithm constrained covalent bond for the equilibration steps. Long range electrostatics was calculated by using Particle Mesh Ewald (PME) method with Fourier grid spacing of 1.4 Å. After equilibration at 1 bar and 300 K under NPT ensemble with a time step of 2 fs, production molecular dynamics simulation was run for 200 ns with the time step defined as 2 fs. Trajectories were saved and XMGRACE was used to analyze results.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The crystal structure of Drosophila melanogaster 3,4-dihydroxyphenylacetaldehyde synthase has been deposited in the Protein Data Bank (PDB) with the PDB ID 6JRL. All required data will be made available upon acceptance of the manuscript.

Acknowledgements

Crystallographic data collection of this work was carried out at the National Synchrotron Light Source, Brookhaven National Laboratory. We are grateful to Dr. Howard Robinson for help with data collection, and to Dr. Dayong Wang for help with data analysis. The research of Qian Han was supported by the National Natural Science Foundation of China (Grant Nos. 31860702; 31472186) and the National High-end Foreign Experts Recruitment Plan (G2021034003L). The research of Chenghong Liao was supported by the National Natural Science Foundation of China (Grant No. 31960703).

Author contributions

JC, CJV, SSW, HC, YT, JL, HG, CL, QH and JYL performed experiments and analyzed data; QH and CL conceived the experiments, and JC, CJV, SSW, CL and QH wrote the manuscript. All authors read and edited the manuscript.

Competing interests

The authors declare that the research of this study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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DatasetS1.xlsx
Supplementary Dataset 1
DHPASSSI230302a.docx
Supplementary Information
DHPAASreportingsummary.pdf
Reporting Summary

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3,4-Dihydroxyphenylacetaldehyde synthase evolved an ordered and distinct active site to promote elastic cuticle formation and blood intake in Aedes aegypti

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Abstract

Figures

Introduction

Results

Abdominal elasticity varies between male and female adult mosquitoes

DHPAAS knockdown increases mortality and reduces blood meal volume

DHPAAS knockdown disrupts the abdominal cuticle

DHPAAS knockdown reduces egg hatching

DHPAAS knockdown alters cuticular structure

Differential gene expression after DHPAAS knockdown

Crystal structure of insect DHPAAS

Structural comparison of DHPAAS and DDC

AeDHPAAS full-length model

Molecular docking and simulation of AeDHPAAS substrate binding

Discussion

Material And Methods

Abdominal injection of adult mosquitoes

RNAi knockdown of DHPAAS in adult mosquitoes

RNAseq and analysis

Transmission electron microscopy

Preparation of recombinant DmDHPAAS

Protein crystallization and structure determination

Building a full-length AeDHPAAS model

Molecular docking and simulation

Declarations

References

Additional Declarations

Supplementary Files

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