Exploring the Structure-Function Relationships in a 5-Aminolevulinic Acid Synthase and the Use of Protein Engineering to Expand its Substrate Range.

Abstract

5-Aminolevulinate synthase (ALAS) is a PLP-dependent enzyme that catalyzes the production of 5-aminolevulinate from succinyl-CoA and glycine. Its ability to catalyze the essentially irreversible C-C bond formation has significant potential in chemoenzymatic synthesis of α-amino ketones. Native ALAS, unfortunately, is extremely substrate-selective, and this seriously limits its synthetic utility. Here, we have used three different protein engineering strategies to overcome this problem for the acyl-CoA substrate. By combining previously reported mutation results and structural analysis, a series of site-saturation mutagenesis/screening efforts were focused on R21, T82, N84, and T362 of Rhodopseudomonas palustris ALAS. These yielded single, double, and triple mutants with significantly improved substrate ranges. The steady-state kinetic parameters of several key variants were determined. These data were analyzed in the framework of the ALAS catalytic mechanism to identify the steps that may have been impacted. The most active variant was used in a larger-scale reaction to demonstrate its synthetic potential. Taken together, our results show how ALAS might become a useful biocatalyst for α-amino ketone synthesis and have also allowed us to comment on the relative merits of each the three protein engineering strategies utilized.