Clarifying the Molecular Mechanisms of Mosquito Cuticle Assembly ¨DNew physiological and molecular views of an essential insect cuticle enzyme¨D

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May 28, 2025

Researchers from the Tokyo University of Agriculture and Technology (TUAT) Graduate School Engineering, Institute of Engineering, Division of Biotechnology and Life Science, in collaboration with researchers at Hainan University, have uncovered two key pieces of evidence that clarify how mosquito cuticle structures are assembled. First, the discovery of potential oxygen-delivery tunnels in the crystal structure of DHPAAS ¹ provides a mechanistic explanation for how this enzyme oxidizes its substrate 3,4-dihydroxyphenylalanine (L-DOPA), to produce the potential cuticle cross-linking agent 3,4-dihydroxyphenylacetaldehyde (DHPAA). Second, solid-state NMR analysis ² detected catechol ring carbons in mosquito cuticle samples, supporting the incorporation of DHPAA-derived products into the cuticle matrix.

Background
The mosquito cuticle, essential for maintaining body structure and survival, is formed through a complex cross-linking process involving specialized enzymes, including DHPAAS, which converts L-DOPA to DHPAA in the cuticle. However, the detailed oxygen-dependent mechanism of DHPAAS has remained unclear, and the incorporation of DHPAA-derived compounds into the insect cuticle has yet to be experimentally confirmed. In this study, the international research team confirmed that DHPAAS plays an essential role in maintaining abdominal integrity and promoting cuticle formation in Aedes aegypti mosquitoes. Furthermore, by solving the first crystal structure of insect DHPAAS, the team identified potential oxygen-delivery tunnels, providing new insights into the oxygen-dependent catalytic mechanism of this essential cuticle enzyme (Figure 1). Solid-state NMR analysis of mosquito cuticle provided supporting evidence for the incorporation of DHPAA-derived compounds, linking DHPAAS activity to cuticle cross-linking at the molecular level (Figure 2).

Research Team
The potential oxygen tunnels were discovered by TUAT Associate Professor Christopher J. Vavricka (shared corresponding and shared first author), who coordinated the overall study with support from the G-7 Scholarship Foundation, the Takeda Science Foundation, and JSPS KAKENHI Grant Number JP25K01588. The crystallographic and mosquito studies were led by Professor Qian Han (corresponding author), Professor Chenghong Liao (corresponding author), and Dr. Jing Chen (shared first author) of Hainan University. Solid-state NMR experiments were led by Professor Yasumoto Nakazawa, with assistance from Professor Keiichi Noguchi, Mr. Yuri Matsumoto, and Associate Professor Vavricka, at the Division of Biotechnology and Life Science, Institute of Engineering, Graduate School of Engineering, TUAT.

Research Results
The crystal structure of insect DHPAAS revealed hydrophobic tunnels with potential to deliver oxygen to pyridoxal 5'-phosphate in the active site; this discovery offers a new mechanism to explain how oxygen participates in the direct conversion of L-DOPA to the putative cross-linking precursor DHPAA. Complementary gene knockdown experiments demonstrated that DHPAAS is essential for mosquito abdominal integrity, cuticle formation and survival. Furthermore, solid-state NMR analysis detected chemical shifts consistent with catechol ring carbons in mosquito cuticles, providing evidence that DHPAA-derived compounds are likely incorporated into the cuticle matrix.

Future Outlook
The research team will continue to investigate the enzymatic and structural mechanisms underlying insect cuticle assembly, focusing on the biochemical processes that enable the formation of highly durable materials. The structural insights provided by this study also offer a new basis for engineering oxygen-dependent enzymatic reactions to produce valuable aromatic compounds. In addition, the findings of the current study may contribute to the development of future mosquito control methods.

Glossary of Terms
1) DHPAAS (3,4-dihydroxyphenylacetaldehyde synthase)
An enzyme that converts L-DOPA into the reactive aldehyde DHPAA, which is believed to participate in cuticle cross-linking reactions in insects.
2) Solid-state NMR (nuclear magnetic resonance) analysis
A method for analyzing molecular structures of complex solid materials such as insect cuticles, where conventional solution NMR or crystallography cannot be easily applied due to heterogeneous composition.

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Christopher J. Vavricka
Associate Professor
Division of Biotechnology and Life Sciences
Institute of Engineering, Graduate School of Engineering
Tokyo University of Agriculture and Technology
E-mail£ºchris(at)go.wxanhx.com