Microbial soluble aromatic prenyltransferases for engineered biosynthesis

Abstract

Prenyltransferase (PTase) enzymes play crucial roles in natural product biosynthesis by transferring isoprene unit(s) to target substrates, thereby generating prenylated compounds. The prenylation step leads to a diverse group of natural products with improved membrane affinity and enhanced bioactivity, as compared to the non-prenylated forms. The last two decades have witnessed increasing studies on the identification, characterization, enzyme engineering, and synthetic biology of microbial PTase family enzymes. We herein summarize several examples of microbial soluble aromatic PTases for chemoenzymatic syntheses of unnatural novel prenylated compounds.

Keywords: Biosynthesis; DHN, dihydroxynaphthalene; DMAPP, dimethylallyl diphosphate; DMATS, dimethylallyltryptophan synthase; DMSPP, dimethylallyl S-thiolodiphosphate; Enzyme engineering; FPP, farnesyl diphosphate; GFPP, geranyl farnesyl diphosphate; GPP, geranyl diphosphate; GSPP, geranyl S- thiolodiphosphate; IPP, isopentenyl pyrophosphate; Microbial prenyltransferase; PPP, phytyl pyrophosphate; PTase, prenyltransferase; Prenylation; RiPP, ribosomally synthesized and posttranslationally modified peptide; Synthetic biology; THN, 1,3,6,8-tetrahydroxynaphthalene.

Fig. 1

Examples of natural products modified by prenyl moieties.

Fig. 2

The crystal structures of (A) ABBA-type PTase NphB (1ZDY), (B) DMATS-type PTase AtaPT (5KCL), (C) UbiA-type PTase ApUbiA (4OD4), and (D) the catalytic pathway and tested substrates for NphB (the prenylated sites are highlighted).

Fig. 3

(A) The substrate specificities of the MpnD, TleC, and TleC mutants. (B) The tested substrates for TleC and MpnD. (C) Superimpositions of the active site residues of TleC (green) and MpnD (cyan) with (−)-indolactam V and DMSPP.

Fig. 4

The catalytic reactions of (A) FtmPT1 and its Y205X mutant with cyclo-L-Trp-L-Pro, and (B) FtmPT1 and its two possible mechanisms with (E)-4-(1H-indol-3-yl)but-3-en-2-one.

Fig. 5

(A) A Cope rearrangement mechanism for the reaction catalyzed by FgaPT2 WT and the K174A mutant. (B) The enzyme reactions of FgaPT2 and its K174F/R244X mutants.

Fig. 6

(A) The substrate promiscuity of AtaPT and the G326 M mutant. (B) Some selected aromatic acceptors for AtaPT (the prenylated sites are highlighted). (C) The apo structure of AtaPT. (D) The complex structures of AtaPT-GSPP and (+)-butyrolactone II.

Fig. 7

(A) The native enzymatic reactions of BrePT, CdpC3PT, and FgaPT2. (B) The geranylated positions catalyzed by the engineered dimethylallyl transferases with gatekeeping residue mutations.

Fig. 8

The prenyltransfer reactions catalyzed by (A) AmbP1 and (B) AmbP3.

Fig. 9

The active sites of (A) AmbP1 with 41 and GSPP, (B) AmbP1 with 41, GSPP, and Mg²⁺, (C) AmbP3 with 44 and DMSPP, and (D) AmbP3 with 5 and DMSPP.

Fig. 10

The structures of kawaguchipeptins A (49) and B (50).

Fig. 11

The proposed catalytic mechanism of KgpF with Fmoc-Trp (51).

Fig. 12

(A) The structure of cyclic-(INPYLYP) (52). (B) The cocrystal structures of PagF (F222 shown), DMSPP, and Mg²⁺.

Fig. 13

The catalytic profiles of AsTPS and FgGS as (A) terpene cyclases (the original function) and (B) prenyltransferases (newly found function).