Biosynthetic Pathways of Alaremycin and Its Derivative: Inhibitors of Porphobilinogen Synthase in Porphyrin Biosynthesis from Streptomyces sp. A012304.

Abstract

The antibiotic alaremycin (5-acetamido-4-oxo-5-hexenoic acid, 1), isolated from Streptomyces sp. A012304, structurally resembles 5-aminolevulinic acid (ALA), a precursor in porphyrin biosynthesis, and inhibits porphobilinogen synthase, the enzyme responsible for catalyzing the first common step of this pathway. In our previous study, the biosynthetic gene cluster responsible for alaremycin production-composed of almA (ALA synthase homologue), almB (N-acetyltransferase), almC (oxidoreductase), and almE (MFS-type transporter)-was identified, and a potential biosynthetic pathway was proposed. In this study, the biosynthetic pathway of 1 was confirmed by detecting intermediates using the liquid chromatography-mass spectrometry/MS (LC-MS/MS) analysis of extracts from Escherichia coli cells transformed with the biosynthetic genes, followed by in vitro reconstitution of the biosynthetic reactions using purified enzymes. AlmA catalyzed the condensation of l-serine and succinyl-CoA to produce 5-amino-6-hydroxy-4-oxohexanoic acid (2), AlmB catalyzed the N-acetylation of 2 to produce 5-acetamido-6-hydroxy-4-oxohexanoic acid (3), and AlmC catalyzed the dehydration of 3 to form 1. The AlmC-catalyzed reaction may involve a two-step mechanism including reduction by NADH and oxidation by Fe³⁺. Additionally, a novel derivative of 1 was identified in the culture broth of the producer strain, and its structure was determined as 5,6-dihydroalaremycin (5-acetamido-4-oxohexanoic acid, 4). It was revealed that 4 is synthesized via the same biosynthetic pathway but with AlmA and AlmB utilizing l-alanine as the amino acid precursor instead of l-serine.