Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Dihydrodipicolinate synthase (DHDPS) is crucial for lysine biosynthesis in bacteria.
• Active DHDPS enzymes were previously thought to be exclusively homo-tetrameric, with dimers exhibiting minimal activity.
• Tetramerization was proposed to stabilize the enzyme by reducing dynamics.

Purpose of the Study:

• To investigate the quaternary structure and catalytic mechanism of DHDPS from methicillin-resistant Staphylococcus aureus (MRSA).
• To determine if the MRSA DHDPS enzyme functions as a dimer, contrary to established models.
• To elucidate the structural and kinetic properties of the MRSA DHDPS dimer.

Main Methods:

• Fluorescence-detected analytical ultracentrifugation to study monomer-dimer equilibrium and dissociation constants.
• Enzyme kinetics assays to determine catalytic parameters and substrate inhibition.
• Near atomic resolution (1.45 Å) X-ray crystallography to confirm dimeric structure.

Main Results:

• MRSA DHDPS exists in a monomer-dimer equilibrium, with tighter dimerization in the presence of pyruvate.
• The dimeric MRSA DHDPS exhibits a ping-pong kinetic mechanism and is insensitive to lysine inhibition.
• The crystal structure reveals an extensive dimerization interface in the MRSA DHDPS dimer.

Conclusions:

• The MRSA DHDPS dimer is catalytically active, challenging the necessity of tetramerization for DHDPS function.
• Substrate binding significantly influences the dimerization state of MRSA DHDPS.
• The extensive dimerization interface suggests evolutionary divergence in DHDPS quaternary structure.