Evidence for benzylsuccinate synthase subtypes obtained by using stable isotope tools

Area of Science:

• Biochemistry
• Enzymology
• Microbial Metabolism

Background:

• Benzylsuccinate synthase (Bss) catalyzes a key reaction involving hydrogen-carbon bond cleavage in toluene metabolism.
• Understanding the Bss reaction mechanism is crucial for elucidating microbial degradation pathways.
• Stable isotope analysis offers powerful tools to probe enzymatic reaction mechanisms.

Purpose of the Study:

• To investigate the reaction mechanism of benzylsuccinate synthase (Bss), focusing on hydrogen-carbon bond cleavage.
• To compare the catalytic mechanisms of Bss enzymes from facultative and obligate anaerobic bacteria.
• To determine if two-dimensional compound-specific stable isotope analysis (2D-CSIA) can differentiate between Bss isoenzymes.

Main Methods:

• Utilized stable isotope tools, including deuterium-labeled toluene, for mechanistic studies.
• Performed in vitro studies with Bss-containing cell extracts and compared with in vivo whole-cell experiments.
• Employed two-dimensional compound-specific stable isotope analysis (2D-CSIA) to measure kinetic isotope effects (Λ values).
• Analyzed the phylogeny of Bss variants through sequence analysis of the BssA gene.

Main Results:

• Λ values from in vitro Bss studies mirrored in vivo data, confirming Bss as the catalyst responsible for observed isotope effects.
• Bss enzymes from facultative anaerobes exhibited smaller Λ values and higher hydrogen exchange probabilities compared to obligate anaerobes.
• Phylogenetic analysis of BssA correlated with observed differences in Λ values and hydrogen exchange, suggesting distinct isoenzyme mechanisms.

Conclusions:

• Subtle differences exist in the reaction mechanisms of Bss isoenzymes from facultative and obligate anaerobes.
• Two-dimensional compound-specific stable isotope analysis (2D-CSIA) can successfully differentiate between these Bss isoenzymes.
• The findings provide insights into the evolution and functional diversity of benzylsuccinate synthase.