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

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 Dfructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-D-[6-2 H 1 ]-and (6R)-D-[6-2 H 1 ]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2 H atom of (6S)-D-[6-2 H 1 ]glucose and no incorporation of the 2 H atom from (6R)-D-[6-2 H 1 ]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-[6-2 H 1 ]-and (6R)-D-[6-2 H 1 ]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.

Inositol lipid synthesis is widespread in host-associated Bacteroidetes

“…We further characterized the BT_1526 protein by determining the X -ray crystal structure to 2.0 Å resolution by molecular replacement using a model derived from the Archaeoglobus fulgidus MIPS structure (PDBID: 3QVT) (refinement statistics in Supplementary Table 1). The overall 3D fold of BT_1526 is consistent with other members of this family with a Rossman fold-like nucleotide binding domain (residues 4-244, 355-429) with an intercalated catalytic/dimerization domain (residues 245-354) 33–36 . The protein adopts a tetrameric dimer of dimers quaternary structure of approximately 188 kDa, with the dimerization domain forming an extended beta-sheet between monomers to create a saddle-like interface for the two dimers within the tetramer.…”

Biosynthesis of cyclitols

“…The crystal structure of Ari2 complexed with NAD + elucidated the active site of Ari2 and NAD + recognition mechanism. 53 The binding mode of NAD + with Ari2 and MIPS appears to be the same. However, how Ari2 accommodates F6P selectively remains unclear.…”

“…54 Ari2 selectively recognizes F6P as substrate and converts it to a ve-membered C6-cyclitol Natural Product Reports Review phosphate according to the MIPS catalytic reaction mechanism. 52,53 The stereochemistry of the deprotonation step to form the enolate intermediate, aldol-type condensation, and reduction of the ketone intermediate in the nal step, is the same as that in the MIPS reaction (Fig. 14).…”

“…13). 52,53 Aristeromycin and its precursor neplanocin A inhibit S-adenosyl-L-homocysteine hydrolase and show potent antivirus activity. 54 Ari2 selectively recognizes F6P as substrate and converts it to a ve-membered C6-cyclitol Natural Product Reports Review phosphate according to the MIPS catalytic reaction mechanism.…”

“…53 Therefore, the pro-R proton on the C-6 methylene group of F6P is deprotonated to give a presumed enolate intermediate prior to C-C bond formation. 53 The only difference between the Ari2 and MIPS reactions is the substrate, with Ari2 recognizing F6P instead of G6P. The crystal structure of Ari2 complexed with NAD + elucidated the active site of Ari2 and NAD + recognition mechanism.…”