Manipulation of prenylation reactions by structure-based engineering of bacterial indolactam prenyltransferases

Abstract

Prenylation reactions play crucial roles in controlling the activities of biomolecules. Bacterial prenyltransferases, TleC from Streptomyces blastmyceticus and MpnD from Marinactinospora thermotolerans, catalyse the 'reverse' prenylation of (-)-indolactam V at the C-7 position of the indole ring with geranyl pyrophosphate or dimethylallyl pyrophosphate, to produce lyngbyatoxin or pendolmycin, respectively. Using in vitro analyses, here we show that both TleC and MpnD exhibit relaxed substrate specificities and accept various chain lengths (C5-C25) of the prenyl donors. Comparisons of the crystal structures and their ternary complexes with (-)-indolactam V and dimethylallyl S-thiophosphate revealed the intimate structural details of the enzyme-catalysed 'reverse' prenylation reactions and identified the active-site residues governing the selection of the substrates. Furthermore, structure-based enzyme engineering successfully altered the preference for the prenyl chain length of the substrates, as well as the regio- and stereo-selectivities of the prenylation reactions, to produce a series of unnatural novel indolactams.

Figure 1. Reaction schemes of indole prenyltransferases.

Reaction schemes for the conversion of (−)-indolactam V (1) into (a) lyngbyatoxin A (2) by S. blastmyceticus TleC and M. producens LtxC, and (b) pendolmycin (3) by M. thermotolerans MpnD.

Figure 2. The in vitro enzyme reactions of S. blastmyceticus TleC and M. thermotolerans MpnD.

HPLC elution profiles of the enzyme reaction products of (a) TleC and (b) MpnD from (−)-indolactam V and prenyl pyrophosphates with various chain lengths as substrates.

Figure 3. Comparison of the substrate binding modes in S. blastmyceticus TleC and M. thermotolerans MpnD.

Closeup views of the active site architectures in (a) the TleC-1-DMSPP ternary complex and (b) the MpnD-1-DMSPP ternary complex. (c) Superimposition of the active site residues of TleC and MpnD. The key three residues discussed here are highlighted by red squares. (−)-Indolactam V (1) and DMSPP are depicted by blue and orange stick models, respectively. The amino acid residues located in the pyrophosphate and indolactam-binding sites are represented by limegreen and green stick models in TleC, and salmon and magenta stick models in MpnD, respectively. The F_o−F_c electron density maps of the 1 and DMSPP are represented as a black mesh, contoured at +2.5σ. Dashed yellow lines represent hydrogen bonds. Red spheres depict the water molecules.

Figure 4. Comparison of the active site structures of S. blastmyceticus TleC and M. thermotolerans MpnD.

Closeup views of the active site cavities of (a) the TleC apo structure and (b) the TleC complex structure with 1 and DMSPP, (c) superimposed view of apo and complex structure of TleC, (d) the MpnD apo structure, (e) the MpnD complex structure with 1 and DMSPP, and (f) superimposed view of apo and complex structure of MpnD. (−)-Indolactam V (1) and DMSPP are depicted by a cyan and blue stick models, respectively. The amino acid residues located in the pyrophosphate and indolactam-binding sites are represented by limegreen and green stick models in TleC, and magenta and salmon stick models in MpnD, respectively. The three important residues discussed in the main text are depicted by cyan stick models. The ‘prenyl binding pocket' in the TleC-1-DMSPP ternary complex structure is highlighted by a red surface. The residues and surface of apo structure and complex structure of TleC are represented by palegreen and salmon, respectively. The residues and surface of apo structure and complex structure of MpnD are represented by pink and pale cyan, respectively.

Figure 5. The in vitro enzyme reactions of the wild-type and mutants of S. blastmyceticus TleC and M. thermotolerans MpnD.

HPLC elution profiles of the enzyme reaction products from (−)-indolactam V and (a) DMAPP or (b) GPP as substrates.

Figure 6. The stereo view of the active site structures of TleC and FtmPT1.

The active site residues of TleC and FtmPT1 from A. fumigatus are shown by green and grey stick models, respectively. The respective substrates 1 and DMSPP in TleC, and brevianamide F and DMSPP in FtmPT1 are represented by blue and brown stick models, respectively. Structures were superposed based on the Cα-atom.