Dissection of the neocarazostatin: a C₄ alkyl side chain biosynthesis by in vitro reconstitution

Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C₄ alkyl side chain attached to ring "A". Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C₄ unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of β-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C₄ alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.