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

Abstract

Prenylated secondary metabolites including indole derivatives usually demonstrate improved biological and pharmacological activities, which make them promising candidates for drug discovery and development. The transfer reactions of a prenyl moiety from a prenyl donor, e.g. dimethylallyl diphosphate (DMAPP), to an acceptor is catalysed by prenyltransferases. One special group of such enzymes uses DMAPP and tryptophan as substrates with dimethylallyltryptophans as reaction products and functions therefore as dimethylallyltryptophan synthases (DMATSs). Sequence homology search with known tryptophan prenyltransferases from Streptomyces led to identification of a putative prenyltransferase gene MolI14.36 in Micromonospora olivasterospora. Expression and biochemical investigations revealed that MolI14.36 acts as a tryptophan C6-prenyltransferase (6-DMATS). Study on substrate specificity of 6-DMATS displayed a significantly high activity towards d-tryptophan, which prompted us to carry out comparative studies on enantioselectivity, regioselectivity and multiple prenylation ability of additional DMATSs including FgaPT2, 5-DMATS, 5-DMATS, 6-DMATS, 6-DMATS and 7-DMATS towards l- and d-isomers of tryptophan and their analogues. The relative activities of the tested enzymes towards d-tryptophan differ clearly from each other. Incubation of l-, d-isomers or the racemates of 5-, 6- and 7-methyltryptophan revealed distinctly different preferences of the DMATS enzymes. Interestingly, 6-DMATS and 5-DMATS accepted 5-methyl-d-tryptophan much better than the l-enantiomer. Furthermore, the conversion yields of the d-isomers were strongly inhibited in the reactions with racemates. More interestingly, the regioselectivities of FgaPT2, 5-DMATS and 7-DMATS towards d-tryptophan and its C5-methylated derivative differed clearly from those of the l-forms. In addition, both mono- and diprenylated products were clearly detected for 5-DMATS with l- and d-enantiomers of tryptophan and their methylated derivatives.

Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1Ff

“…Since studies on other DMATS showed substrate promiscuity, − the substrate scope of DMATS1 Ff was surveyed by testing different trypthophan derivatives and structurally related compounds (Figure S6). For substrates missing either the free amino group or the free carboxylic acid group no product formation was observed.…”

One-Pot Biocatalytic Synthesis of Substituted d-Tryptophans from Indoles Enabled by an Engineered Aminotransferase

“…The engineering of an aminotransferase displaying near-native catalytic efficiency toward d -tryptophan (DAAT-V33G/T242G) was key to improve the performance of this procedure, which compensates for the unavailability of a d -selective TrpS. Many of the substituted d -tryptophans that we synthesized in preparative scale using our one-pot procedure are useful synthetic building blocks for a broad range of target molecules such as mitragynine and related 9-methoxy-substituted indole alkaloids (from d - 2d ), prenylated tryptophans (from d - 2f and d - 2i ), inhibitors of breast cancer resistance protein (from d - 2j ), and necrostatins (from d - 2k ). The biocatalytic approach developed here allows for the efficient synthesis of useful chiral synthons for the preparation of complex natural products and pharmaceutical ingredients.…”

Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases

“…IptA, 5-DMATS Sc , and 6-DMATS Mo preferred DMAPP as a cosubstrate. 61,63,64,115,122 Of the three, only 6-DMATS Mo accepted GPP as a cosubstrate, but with a low turnover rate. 64 In contrast, PriB was reported to incorporate GPP with a turnover rate near 100%, demonstrating that the CPDR was not the only structural factor determining the cosubstrate scope, if at all.…”

“…Whereas DKP DMATSs can accommodate relatively large substrates (i.e., DKPs, naphthalenes, and aszonalenins), Trp DMATSs primarily prenylate simple indole derivatives, although FgaPT2 and PriB are notable exceptions (Table 1). 54,58,59,[61][62][63]65,[114][115][116][117][118][119] These two Trp DMATSs prenylated the indole moiety of the large antibiotic daptomycin, the structural basis of which has not yet been fully dened. 65 The ability of PriB, FgaPT2, and CdpNPT (the only three DMATSs shown to modify daptomycin) to generate prenylated daptomycin analogues provides a convenient starting point for the discussion of the steric constraints of the binding pockets of Trp DMATSs relative to those of DKP DMATSs.…”