A KAS III-like Enzyme and an Amidohydrolase in Nataxazole Biosynthesis Direct Formation of the Benzoheterocycle Moiety

Benzoxazole is an important heterocycle building block frequently found in medicinal compounds and naturally occurring alkaloids. Deciphering the biosynthetic machinery of benzoxazole would facilitate the efficient synthesis of benzoxazole-based drugs. Nataxazole represents a unique alkaloid class containing two benzoxazole moieties (A and B). Previous studies have established the enzymatic mechanism underlying the biosynthesis of benzoxazole moiety A (fusion of two 3-hydroxyanthranilic acid molecules). Here, we report that the same biosynthetic machinery of nataxazole employs a different logic, which involves a 3-oxoacyl-ACP synthase III-like enzyme (NatS), to assemble benzoxazole moiety B (fusion of 3-hydroxyanthranilic acid with 6-methylsalicylic acid). Biochemical and structural characterization indicated that NatS catalyzes a transacylation reaction to generate an unstable ester intermediate, which immediately undergoes cyclization and dehydration catalyzed by the amidohydrolase NatAM to give benzoxazole moiety B. Interestingly, NatS can also catalyze C–N and C–S bond formation and exhibits remarkable substrate promiscuity, inspiring us to synthesize various benzoheterocycles following the assembly logic of moiety B. Together with our investigation into the biosynthesis of nataxazole analogue A33853, we further identified two key amino acids responsible for NatAM’s substrate recognition to generate two benzoxazole moieties in nataxazole. Finally, we successfully developed a chemoenzymatic approach to produce transthyretin stabilizer Tafamidis (Vyndamax). Therefore, our study found the missing piece of the benzoxazole biosynthesis puzzle, providing a promising strategy to create diverse types of benzoheterocycle compounds.