Cys139Ser mutation in dimeric nucleoside diphosphate kinase generates catalytically competent monomer

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Nucleoside diphosphate kinase (NDK) enzymes are crucial for cellular metabolism.
• NDKs from various organisms exhibit diverse quaternary structures, including hexamers and tetramers.
• Halophilic NDKs, such as HaNDK and CsNDK, have been observed to form dimeric structures.

Purpose of the Study:

• To investigate the structural and functional significance of the dimeric form of halophilic NDK from Chromohalobacter salexigens (CsNDK).
• To explore the role of a conserved cysteine residue (Cys139) at the monomer-monomer interface in CsNDK stability and activity.

Main Methods:

• Field flow fractionation was employed to assess the oligomeric state of wild-type and mutant CsNDK.
• Circular dichroism (CD) spectroscopy was used to evaluate protein secondary structure.
• Protease susceptibility assays using endoproteinase GluC were performed to probe structural integrity.
• Enzymatic activity assays were conducted to determine the catalytic function of monomeric and dimeric forms.

Main Results:

• CsNDK was confirmed to exist as a dimer, similar to HaNDK.
• Mutation of the conserved Cys139 to serine resulted in the dissociation of CsNDK into monomers in buffer.
• Circular dichroism profiles indicated no significant change in secondary structure between wild-type and mutant CsNDK.
• The Cys139Ser mutant exhibited increased susceptibility to protease cleavage, which was mitigated by the presence of the NDK substrate ATP.
• The monomeric form of CsNDK retained enzymatic activity, though the dimeric form might be the preferred active state in the presence of substrates.

Conclusions:

• The conserved Cys139 residue plays a critical role in maintaining the dimeric structure of CsNDK.
• While CsNDK can function as a monomer, substrate binding (ATP) may promote or stabilize the dimeric active conformation.
• These findings shed light on the structural plasticity and substrate-dependent quaternary organization of halophilic nucleoside diphosphate kinases.