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

Carla Araya-Cloutier,Bianca Martens,Gijs Schaftenaar,Franziska Leipoldt,Jean-Paul Vincken,Harry Gruppen,Emily J Parker

doi:10.1371/journal.pone.0174665

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.

Highlights

Prenylation is one of nature’s tools to modulate the bioactivity of primary [1] and secondary metabolites [2, 3] by increasing their lipophilicity and, thereby, their interactions with biological targets, such as proteins and membranes [4]
Activity of Streptomyces roseochromogenes CloQ (SrCloQ) was confirmed in control experiments with its genuine substrate (4-HPP); incubations without SrCloQ did not yield prenylated products (Figure D in S1 File)
The structures of compounds are oriented with the B-ring of the phenolics in the same direction as the phenol ring of the genuine substrate 4-HPP

Summary

Introduction

Prenylation is one of nature’s tools to modulate the bioactivity of primary [1] and secondary metabolites [2, 3] by increasing their lipophilicity and, thereby, their interactions with biological targets, such as proteins and membranes [4]. Aromatic PTs catalyse the transfer reaction of prenyl moieties onto aromatic acceptors, such as phenolic acids, (iso)flavonoids, coumarins, naphthalenes, phenazines, and indole derivatives. These enzymes contribute substantially to the large diversity of secondary metabolites present in plants, fungi, and bacteria [6, 7]. PTs responsible for the production of glyceollins in soybean (Glycine max L.) and phaseollin in kidney bean (Phaseolus vulgaris L.) are localized in the membrane of plastids. Solubilisation of these PTs required detergents, which negatively affected enzyme activity and stability [10]. An overview of recently characterized plant PTs can be found elsewhere [12]

Methods

Results

Discussion

Conclusion