Structural basis for non-genuine phenolic acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase CloQ from the ABBA/PT-barrel superfamily

Abstract

Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.

Fig 1. Schematic representation of the proposed mechanism of prenylation by SrCloQ, adapted from previous work [17].

Important residues interacting with the donor and acceptor substrate are shown in grey colour. The donor substrate DMAPP is shown in red, whereas the genuine substrate is shown in blue.

Fig 2. Aromatic acceptor substrates and their percentage of conversion (molar %) by SrCloQ.

The atoms shared with the genuine substrate are highlighted in blue colour. Phenolic substrates with green label represent the best acceptor substrates, whereas those with red labels were not utilized by the enzyme.

Fig 3. SrCloQ prenylated equol products.

RP-UHPLC-UV (A) and MS in NI (B) profiles of equol and prenylated equol products (products 8 and 9 in Table 1) after incubation of equol with DMAPP and SrCloQ. MS² spectra of C-prenylated equol (m/z 309) at retention times 16.9 (8, panel C) and 17.1 min (9, panel D). The proposed RDA fragmentation pathways are shown as inset.

Fig 4. SrCloQ prenylated 4’-hydroxy-7-methoxyflavone products.

RP-UHPLC-UV (A) and MS in PI (B) profiles of 4’-hydroxy-7-methoxyflavone and prenylated products (14, 15 and 16 in Table 1) after incubation of 4’-hydroxy-7-methoxyflavone with DMAPP and SrCloQ. MS² spectra of C-prenylated isomer (m/z 359, Na adduct) at retention time 17.1 min (15, panel C) and O-prenylated isomer at 18.1 min (16, panel D). The proposed RDA fragmentation pathway is shown as inset.

Fig 5. Molar composition of the prenylated products obtained with SrCloQ.

Error bars represent the standard deviation; n.a., not annotated.

Fig 6. Phenolic aromatic substrates docked in the active site of SrCloQ (PDB 2XLQ).

(A) Comparison of equol with the A-ring (light blue) and B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-Dihydroxyflavone (green) and daidzein (dark red) with their B-ring towards the prenyl donor. Protein surface is coloured according to the lipophilic potential using MOE software default’s setting: yellow being lipophilic and blue hydrophilic (cut-off of 2.5). Donor substrate DMAPP is shown in gray with the phosphate group in pink. Residues that interact with the acceptor substrates are shown in orange: Glu281 anchors the substrate by H-bonding with the phenolic hydroxyl group; Arg160 stabilizes binding by H-bonding with the carbonyl or ether group of the C-ring; Tyr233 aromatic ring can make H-π bonds or π stacking interactions with the aromatic rings of the phenolic substrates, while its carbonyl group can make H-bonding with phenolic hydroxyl groups; Cys297 thiol group can interact with the carbonyl group of the phenolic substrate.