Exploring the\nStructure–Function Relationships\nin a 5‑Aminolevulinic Acid Synthase and the Use of Protein\nEngineering to Expand its Substrate Range

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