Comparison of <i>Alicyclobacillus acidocaldarius</i> <i>o</i>‑Succinylbenzoate Synthase to Its Promiscuous <i>N</i>‑Succinylamino Acid Racemase/<i>o</i>‑Succinylbenzoate Synthase Relatives
Studying
the evolution of catalytically promiscuous enzymes like
those from the <i>N</i>-succinylamino acid racemase/<i>o</i>-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal
mechanisms by which new functions evolve. Some enzymes in this subfamily
have only OSBS activity, while others catalyze OSBS and NSAR reactions.
We characterized several NSAR/OSBS subfamily enzymes as a step toward
determining the structural basis for evolving NSAR activity. Three
enzymes were promiscuous, like most other characterized NSAR/OSBS
subfamily enzymes. However, <i>Alicyclobacillus acidocaldarius</i> OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable
NSAR activity. Competitive inhibition and molecular modeling show
that AaOSBS binds <i>N</i>-succinylphenylglycine with moderate
affinity in a site that overlaps its normal substrate. On the basis
of possible steric conflicts identified by molecular modeling and
sequence conservation within the NSAR/OSBS subfamily, we identified
one mutation, Y299I, that increased NSAR activity from undetectable
to 1.2 × 10<sup>2</sup> M<sup>–1</sup> s<sup>–1</sup> without affecting OSBS activity. This mutation does not appear to
affect binding affinity but instead affects <i>k</i><sub>cat</sub>, by reorienting the substrate or modifying conformational
changes to allow both catalytic lysines to access the proton that
is moved during the reaction. This is the first site known to affect
reaction specificity in the NSAR/OSBS subfamily. However, this gain
of activity was obliterated by a second mutation, M18F. Epistatic
interference by M18F was unexpected because a phenylalanine at this
position is important in another NSAR/OSBS enzyme. Together, modest
NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299
indicate that additional sites influenced the evolution of NSAR reaction
specificity in the NSAR/OSBS subfamily