Catalytic site cooperativity in dimeric methylthioadenosine nucleosidase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs) are bacterial enzymes crucial for quorum sensing and menaquinone synthesis.
• MTANs function as homodimers with catalytic sites located at the dimer interface.

Purpose of the Study:

• To investigate the ligand interactions and cooperative mechanisms within the Staphylococcus aureus MTAN (SaMTAN) homodimer.
• To elucidate the functional roles of the two catalytic sites and their interplay.

Main Methods:

• Enzyme kinetics and thermodynamic analysis of SaMTAN.
• Ligand binding studies using a transition-state analogue (MT-DADMe-ImmA).
• Cysteine reactivity assays and pre-steady-state kinetic experiments.

Main Results:

• SaMTAN exhibits negative catalytic cooperativity, with unequal ligand binding to the two catalytic sites.
• Thermodynamic data indicate distinct binding properties for each site.
• Cysteine reactivity and heterodimer experiments suggest sequential catalysis and product release linked to inter-subunit loop motion.

Conclusions:

• The two catalytic sites in SaMTAN function sequentially, demonstrating negative cooperativity.
• Product release is a rate-limiting step, coupled with conformational changes involving catalytic site loops from neighboring subunits.