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

Biochemistry. 2019 Dec 24;58(51):5112-5116. doi: 10.1021/acs.biochem.9b00981. Epub 2019 Dec 13.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / biosynthesis
  • Adenosine / chemistry
  • Models, Molecular
  • Myo-Inositol-1-Phosphate Synthase / chemistry
  • Myo-Inositol-1-Phosphate Synthase / metabolism*
  • Protein Conformation
  • Stereoisomerism
  • Streptomyces / enzymology

Substances

  • aristeromycin
  • Myo-Inositol-1-Phosphate Synthase
  • Adenosine

Supplementary concepts

  • Streptomyces citricolor