Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domains via diffusible, synthetic substrates

Abstract

Polyketide synthase (PKS) β-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of β-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry. In vitro product isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH as trans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the β-position as d-configured substrates were processed more than 100 times more efficiently than l-alcohols. Unexpectedly, the k_cat/K_M values were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination is predicted to be driven by a combination of allylic A^1,3 strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.

Fig. 1. The modular PKS of tylactone (1). The module 3 β-processing domains and their postulated product are highlighted in red.

Fig. 2. Native and synthetic TylKR3 substrates with their possible β-processing products. The truncated region of the native substrate serving as the basis of substrates 4 and 5 is highlighted in blue.

Scheme 1. Exemplary synthesis of thioether 7b.

Scheme 2. Synthetic route to ketoreductase and thioester substrates 4, 5, and 6b.

Fig. 3. LC-MS/MS traces of in vitro ketoreduction and dehydration reactions. Overnight incubation conducted with KR substrates 4 (panel A) and 5 (panel B) and TylDH3-KR3 in the presence of NADPH. The identity of the β-hydroxy products (shown in blue) was confirmed by co-injection with authentic standards. Incubation with synthetic 7 (panel C) and 9 (panel D) resulted in sole formation of dehydration products 10 and 11 (trace shown in red), respectively. Panels A and C blue trace represents MRM (m/z 340 → 184) and red trace represents MRM (m/z 300 → 181). Panels B and D blue trace represents MRM (m/z 368 → 212) red trace represents MRM (m/z 328 → 151).