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

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.