Stereochemistry in the Reaction of the myo-Inositol Phosphate Synthase Ortholog Ari2 during Aristeromycin Biosynthesis

The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-²H₁]- and (6R)-d-[6-²H₁]­glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the ²H atom of (6S)-d-[6-²H₁]­glucose and no incorporation of the ²H atom from (6R)-d-[6-²H₁]­glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD⁺) to generate the enolate intermediate, which then attacks the C2 ketone to form the C–C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-²H₁]- and (6R)-d-[6-²H₁]­F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD⁺ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.