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Rhipicephalus microplus triosephosphate isomerase dimer interface is stabilized by a key cysteine residue.

Luiz Saramago1, Nallely Cabrera2, Beatriz Aguirre2

  • 1Laboratório Integrado de Bioquímica Hatisaburo Masuda / NUPEM, Laboratório de Bioquímica de Artrópodes Hematófagos/ IBqM, Laboratório de Tecido Conjuntivo/ HUCCF and Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

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|September 14, 2025
PubMed
Summary

Investigating a non-conserved cysteine in Rhipicephalus microplus triosephosphate isomerase (RmTIM) revealed its crucial role in enzyme stability and function. Mutating this residue significantly impairs catalytic efficiency and protein structure, offering potential targets for acaricide development.

Keywords:
Chemical susceptibilityCysteine 86Rhipicephalus microplusSite-directed mutagenesisThermal stabilityTickTriosephosphate isomerase

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Area of Science:

  • Biochemistry and Molecular Biology
  • Enzymology
  • Structural Biology

Background:

  • Triosephosphate isomerase (TIM) is a crucial enzyme in glycolysis.
  • The Rhipicephalus microplus triosephosphate isomerase (RmTIM) is a potential target for acaricide development.
  • The functional role of non-conserved residues, like cysteine at position 86 (C86), in RmTIM's structure and function remains largely unexplored.

Purpose of the Study:

  • To investigate the functional significance of the non-conserved cysteine residue (C86) in RmTIM.
  • To elucidate the structural and kinetic impacts of mutating C86 on RmTIM.
  • To explore the potential of targeting C86 for the development of novel acaricidal compounds.

Main Methods:

  • Site-directed mutagenesis was employed to substitute C86 with aspartic acid (C86D), lysine (C86K), and alanine (C86A).
  • Enzymatic parameters (Vmax, Km, kcat, kcat/Km) were analyzed kinetically.
  • Protein thermal shift (TS) assays and chemical denaturation (guanidine hydrochloride) were used to assess thermodynamic stability.
  • Computational structural analyses, including free energy calculations (ΔG), were performed to understand molecular mechanisms.

Main Results:

  • Mutations C86D and C86K significantly altered kinetic parameters, reducing maximal velocity and catalytic efficiency.
  • All mutants (C86D, C86K, C86A) exhibited substantial thermodynamic destabilization, with melting temperatures (Tm) reduced by up to 27.1 °C.
  • The C86K mutant showed the greatest susceptibility to unfolding and diminished dimeric interface stability (ΔG = -16.0 kcal/mol) compared to wild-type RmTIM (ΔG = -21.2 kcal/mol).
  • Computational analysis predicted disruption of a salt bridge in C86K, compromising dimeric interface integrity.

Conclusions:

  • The non-conserved C86 residue plays a critical structural role in maintaining RmTIM's interfacial integrity and catalytic function.
  • Mutations at C86, particularly C86K, lead to significant destabilization and impaired enzymatic activity.
  • Targeting structural vulnerabilities associated with non-conserved residues like C86 presents a promising strategy for designing specific acaricidal agents against RmTIM.