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.

Structural Basis for the Prenylation Reaction of Carbazole‐Containing Natural Products Catalyzed by Squalene Synthase‐Like Enzymes

“…Catalytic bases for deprotonation have been proposed for aromatic PTs. For example, CloQ from Streptomyces roseochromagenes is thought to neutralize the prenylated σ‐complex by proton abstraction via E281 [21b, 31] . In FgaPT2 from Aspergillus fumigatus , K174 has been proposed to abstract a proton [10] .…”

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB

“…The C1′-C5′ of GSPP are colored yellow, while the C6′-C10′ are colored magenta NphB (Kuzuyama et al 2005), one could anticipate how engineering specific residues within the active site alters the donor specificity with an acceptor of interest, thereby introducing activity with a preferred donor. However, such applications are not limited to NphB; PTs at large are known for their promiscuity toward acceptors (Araya-Cloutier et al 2017;Awakawa and Abe 2019;Elshahawi et al 2017;Schuller et al 2012;Wunsch et al 2015). Therefore, the underlying concepts of acceptordependent donor specificity established for NphB most likely apply to the enzyme class as a whole.…”

“…For example, the accepted prenyl groups can vary in size (5-carbon dimethyallyl, 10-carbon geranyl, 15-carbon farnesyl, 20carbon geranylgeranyl) between PTs, while the attachment can occur from either C1′ or C3′ of the prenyl donors onto various positions of the aromatic acceptors, resulting in normal or reverse prenylation, respectively (Giessen and Marahiel 2015;Winkelblech et al 2015a). Furthermore, PTs have been shown to catalyze the formation of C-C (Araya-Cloutier et al 2017;Bandari et al 2019;Elshahawi et al 2017;Fan et al 2015b;Haagen et al 2007;Winkelblech et al 2015b), C-O (Bandari et al 2017;Haagen et al 2007; Bryce P. Johnson and Erin M. Scull contributed equally to this work.…”

Biotechnological Advances in Resveratrol Production and its Chemical Diversity

“…Such prenylated stilbenoids have shown very effective protective activities in human diseases as well [153]. Similar prenylated resveratrol compounds at the position 3′-, were also with the enzyme NovQ, obtained from Streptomyces spheroids [154,155]. Additionally, the enzymes CloQ and Orf2, obtained from Streptomyces roseochromogeus and Streptomyces sp.…”