Role of an Active Site Loop in the Promiscuous Activities
of <i>Amycolatopsis</i> sp. T‑1-60 NSAR/OSBS
- Publication date
- Publisher
Abstract
The <i>o</i>-succinylbenzoate synthase (OSBS) family
is part of the functionally diverse enolase superfamily. Many proteins
in one branch of the OSBS family catalyze both OSBS and <i>N</i>-succinylamino acid racemization in the same active site. In some
promiscuous NSAR/OSBS enzymes, NSAR activity is biologically significant
in addition to or instead of OSBS activity. Identifying important
residues for each reaction could provide insight into how proteins
evolve new functions. We have made a series of mutations in <i>Amycolatopsis</i> sp. T-1-60 NSAR/OSBS in an active site loop,
referred to as the 20s loop. This loop affects substrate specificity
in many members of the enolase superfamily but is poorly conserved
within the OSBS family. Deletion of this loop decreased OSBS and NSAR
catalytic efficiency by 4500-fold and 25,000-fold, respectively, showing
that it is essential. Most point mutations had small effects, changing
the efficiency of both NSAR and OSBS activities <10-fold compared
to that of the wild type. An exception was F19A, which reduced <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub><sup>OSBS</sup> 200-fold and <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub><sup>NSAR</sup> 120-fold. Mutating the surface residue
R20E, which can form a salt bridge to help close the 20s loop over
the active site, had a more modest effect, decreasing <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> of OSBS and NSAR reactions
32- and 8-fold, respectively. Several mutations increased <i>K</i><sub>M</sub> of the NSAR reaction more than that of the
OSBS reaction. Thus, both activities require the 20s loop, but differences
in how mutations affect OSBS and NSAR activities suggest that some
substitutions in this loop made a small contribution to the evolution
of NSAR activity, although additional mutations were probably required