Fig. 5.

Reprogramming first-ring cyclization regiospecificity by site-directed mutagenesis. (A) Proposed mechanism of regiospecific cyclizations catalyzed by the PT_AtCURS2 and PT_CcRADS2 domains. Enolate formation by deprotonation of C8 (AtCURS2) or C2 (CcRADS2) is promoted by the histidine catalytic base that is polarized by a conserved glutamic acid (AtCURS2: H¹³⁰⁸ and E¹⁴⁹⁷; CcRADS2: H¹³²⁵ and E¹⁵²⁰). The substrates are proposed to thread through alternate routes as directed by different gating residues at the entrance of the cyclization chamber (AtCURS2: W¹⁵⁸⁴; CcRADS2: L¹⁶⁰⁹) and further oriented by an inverted hydrophobic/H-bond donor residue pair lining the rear end of the cyclization chamber (AtCURS2: F¹⁴⁵⁵ and Y¹⁵⁷⁶; CcRADS2: Y¹⁴⁷⁸ and F¹⁶⁰¹). (B) Product profiles (HPLC traces recorded at 300 nm) of S. cerevisiae BJ5464-NpgA (13, 34) cotransformed with YEpAtCURS1 and the YEpATCURS2 derivatives encoding the indicated PT_AtCURS2 domain mutations in AtCURS2.