Functional Characterization and Protein Engineering of <i>Salvia miltiorrhiza</i> Rosmarinic Acid Synthase

Rosmarinic acid (RA) has been incorporated in various nutritious and health-promoting products. Rosmarinic acid synthase (RAS) is a committed enzyme in RA biosynthesis. However, SmRASs from <i>Salvia miltiorrhiza</i> remains functionally uncharacterized. Herein, a group of SmRASs was identified from the <i>S. miltiorrhiza</i>, and recombinant SmRAS1 was demonstrated to convert caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid to form RA. SmRAS1 exhibited maximum activity at 45 °C and pH 8.0 with <i>K</i>_m values of 18 μM and 1647 μM for caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid, respectively. Subsequently, three highly catalytic activity SmRAS1 mutations R367W, G293I, and G293Rwere obtained, exhibiting 1.53-2.15 fold higher catalytic efficiency than the wild-type enzyme. Structural analysis and molecular dynamics simulations revealed that enlarged substrate access tunnel and reduced flexibility of residues^286-295 located above the substrate pocket enhanced its catalytic activity. These results elucidate the mechanism underlying RA biosynthesis in <i>S. miltiorrhiza</i> and provide a promising RAS for RA biosynthesis in microbes.