Main content area

Distinct mechanisms of substrate selectivity in the DRE-TIM metallolyase superfamily: A role for the LeuA dimer regulatory domain

Chen, Wen, Frantom, Patrick A.
Archives of biochemistry and biophysics 2019 v.664 pp. 1-8
Methanococcus, active sites, catalytic activity, coevolution, enzymes, gel chromatography, isotopes, proteins
The use of modular domains in proteins affords nature a simple route to the diversification of protein function, but co-evolution between domains can complicate large-scale functional annotation. The LeuA dimer regulatory domain is primarily responsible for allosteric feedback inhibition of the enzymes isopropylmalate synthase (IPMS) and citramalate synthase (CMS). In addition to this regulatory role, presence of the domain may also affect substrate selectivity in certain members of the enzyme family. To assess the role of the LeuA dimer regulatory domain in substrate selectivity, truncated versions of IPMS and CMS from Methanococcus jannaschii (MjIPMS and MjCMS, respectively) have been created that lack the LeuA dimer regulatory domain. In the case of MjIPMS, loss of the regulatory domain does not affect substrate selectivity, consistent with previous reports identifying conserved active site residues that play this role. Loss of the regulatory domain in MjCMS, however, results in increased functional promiscuity. Both truncated enzymes exhibit a shift in quaternary structure from tetrameric to monomeric forms as judged by size-exclusion chromatography. Kinetic isotope effects reveal that loss of the regulatory domain results in unique effects on catalysis with chemistry becoming more rate-determining in MjIPMS and less rate-determining in MjCMS. Finally, substitution of conserved active site residues in the promiscuous truncated MjCMS affect substrate selectivity while identical substitutions cause no changes in the wild-type enzyme. Overall, the data predicts a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains.