Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme.

Area of Science:

• Biochemistry
• Microbiology
• Molecular Biology

Background:

• Cobamides, vitamin B12 analogs, are essential enzyme cofactors produced by microbes but utilized across all life domains.
• The functional differences arising from cobamide structural diversity remain poorly understood.
• Enzyme selectivity for specific cobamides is critical for microbial metabolism and physiology.

Purpose of the Study:

• To investigate how structural variations in cobamides affect the binding and catalysis of methylmalonyl-CoA mutase (MCM).
• To correlate in vitro cobamide binding selectivity with in vivo cobamide dependence in bacteria.
• To elucidate the biochemical basis of cobamide preferences in microorganisms.

Main Methods:

• In vitro analysis of cobamide binding affinity and catalytic activity with methylmalonyl-CoA mutase (MCM).
• Comparison of MCM orthologs from different bacterial species.
• Bacterial growth assays to assess in vivo cobamide dependence.

Main Results:

• Small changes in the lower ligand of cobamides significantly altered MCM binding affinity.
• Bacterial MCM orthologs exhibited distinct cobamide binding selectivities.
• Catalysis by MCM was less sensitive to lower ligand variations compared to binding.
• In vitro binding selectivity strongly correlated with in vivo cobamide-dependent growth in *Sinorhizobium meliloti*.

Conclusions:

• Enzyme binding selectivity, rather than catalysis, is the primary determinant of cobamide requirements in bacteria.
• Cobamide structural diversity influences bacterial physiology through specific enzyme-cofactor interactions.
• Understanding cobamide selectivity is vital for comprehending microbial interactions in various environments.