Tryptophan C5‐, C6‐ and C7‐Prenylating Enzymes Displaying a Preference for C‐6 of the Indole Ring in the Presence of Unnatural Dimethylallyl Diphosphate Analogues

Abstract

The behavior of four dimethylallyltryptophan synthases (DMATSs) (5‐DMATS and 5‐DMATSSc as tryptophan C5‐prenyltransferases, and 6‐DMATSSa and 6‐DMATSSv as C6‐prenyltransferases) and one L‐tyrosine prenyltransferase with a tryptophan C7‐prenyltransferase activity was investigated in the presence of two unnatural alkyl donors (methylallyl and 2‐pentenyl diphosphate) and one benzyl donor (benzyl diphosphate). Detailed biochemical investigations revealed the acceptance of these dimethylallyl diphosphate (DMAPP) analogues by all tested enzymes with different relative activities. Enzyme products with the allyl or benzyl moiety attached to different positions were identified in the reaction mixtures, whereby C‐6 alkylated or benzylated L‐tryptophan was found as one of the main products. This observation demonstrates a preference of the five prenyltransferases toward C‐6 of the indole ring in the presence of unnatural DMAPP derivatives. Molecular dynamics simulation experiments with a homologous model of 5‐DMATS explained well its reactions with methylallyl and 2‐pentenyl diphosphate. Furthermore this study expands significantly the potential usage of tryptophan prenylating enzymes as biocatalysts for Friedel–Crafts alkylation.magnified image

Characterisation of 6-DMATSMofrom Micromonospora olivasterospora leading to identification of the divergence in enantioselectivity, regioselectivity and multiple prenylation of tryptophan prenyltransferases

“…The observed substrate preferences of the DMATSs differed clearly from each other ( Table 2). As reported previously 20,25,26,28 and demonstrated in this study, 6 was a poor substrate for the C5-prenyltransferases 5-DMATS and 5-DMATS Sc and the C6-methylated derivative 7 was accepted by the C6-prenyltransferases 6-DMATS Sa and 6-DMATS Mo with low conversion yields. Higher conversion yields were found for their orthologue 6-DMATS Sv ( Table 2).…”

“…To gain deeper insights into the substrate preferences of DMATSs towards the stereoisomers of tryptophan, FgaPT2, 5-DMATS, 5-DMATS Sc , 6-DMATS Sa , 6-DMATS Sv , 6-DMATS Mo , and 7-DMATS were overproduced in E. coli and purified to near homogeneity as reported previously. 20,[25][26][27][28] As aforementioned, these enzymes use the same substrates 1a and DMAPP, but catalyse prenylations at different positions of the indole ring. By size exclusion chromatography, these enzymes were determined to have different quaternary structures.…”

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

“…The enzyme's utility as a biocatalyst could likely be expanded considering the wider promiscuities of PTs with respect to their alkyl-donors. Recent studies, including those with the tyrosine-O-PT SirD and the tryptophan C4-PT FgaPT2, have revealed the ability of PTs to transfer a wide array of non-native alkyl-groups onto both native and non-native aromatic acceptors (Bandari et al 2019;Bandari et al 2017;Liebhold et al 2012;Winkelblech et al 2015b;Yu et al 2015). These types of late-stage functionalization reactions on aromatic rings are usually considered difficult to achieve with traditional synthetic chemistry (Hong 2014).…”

“…With this limitation in mind, the final library of 66 native and non-native alkyl-PP analogs, roughly demarcated between allylic (1-44) and aromatic (45-66) groups, was synthesized to explore as large of a chemical space as was feasible. The allylic analogs were designed either to match the natural prenyl donors dimethylallyl pyrophosphate (DMAPP, 2), geranyl pyrophosphate (GPP, 32), and farnesyl pyrophosphate (FPP, 42) (Winkelblech et al 2015a) or to resemble them with variations in alkylchain length, electronic character, and the presence or positioning of branching methyl groups. The resulting library included aliphatic analogs published previously (1, 4-7, 9, 11-13, 17, 18, 20, 24-26, 37) (Bandari et al 2019;Bandari et al 2017), novel GPP derivatives with various terminal groups (35,36,40,41,43,44), and new alkyl-PP analogs with chain lengths between DMAPP and GPP (21-23, 31).…”

“…This reaction scheme, delivers a high level of chemical diversity among the products of PT reactions arising from a combination of factors (Kumano et al 2008;Kuzuyama et al 2005;Shindo et al 2011;Tanner 2015;Tello et al 2008). 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.…”