The biochemical basis for stereochemical control in polyketide biosynthesis

Abstract

One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.

Figure 1

Modular organization (a) the 6-deoxyerythronolide B (6-dEB, 1) synthase (DEBS), (b) the picromycin/methymycin (10-deoxymethynolide (2)/narbonolide (3)) synthase (PICS), and (c) the tylactone (4) synthase (TYLS). Only the first two modules and the loading domains of PICS and TYLS are illustrated. In addition to the three core catalytic domains – the β-ketoacyl-ACP synthase (KS), the acyltransferase (AT), and the acyl carrier protein (ACP) domains – individual extension modules carry varying combinations of tailoring ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains. Loading didomains or tridomains prime module 1 of each synthase with the propionyl starter unit and a thioesterase (TE) domain at the C-terminus of the furthest downstream module catalyzes release and cyclization of the full-length polyketide give the parent macrolide aglycone. Dedicated oxygenases, glycosyl transferases, and methyl transferases then generate the mature antibiotic.

Figure 2

Chiral GC-EI-MS (XIC at m/z 88) of the mixture of (±)-2-methyl-3-hydroxypentanoate methyl esters and retention times of the individual diastereomers (7a-Me, 7b-Me, 8a-Me, and 8b-Me).

Figure 3

Chiral GC-EI-MS (XIC at m/z 88) of methyl (2S,3R)-2-methyl-3-hydroxypentanoate (7a-Me, ret. time 35.04 min) produced by incubation of propionyl-SNAC with DEBS [KS1][AT1], DEBS ACP1 and DEBS KR1 in the presence of methylmalonyl-CoA and NADPH.

Scheme 1

Proposed pathways for methyl group epimerization. In Pathway A, epimerization follows KS-catalyzed condensation, with the KR domain reducing only the correspond diastereomer of the 2-methyl-3-ketoacyl-ACP intermediate. In Pathway B, KS-catalyzed epimerization precedes chain elongation, with the KR domain reducing the resulting intermediate of the correct configuration.

Scheme 2

Stereochemistry of triketide lactone formation catalyzed by dissected DEBS [KS][AT] and ACP domains in combination with recombinant KR domains.

Scheme 3

Stereochemistry of KR-catalyzed reduction of 2-methyl-3-ketoacyl-ACP intermediates generated by combinations of PKS [KS][AT] and ACP domains, as determined by chiral GC-MS analysis of the resulting diketides or triketides. a. Use of propionyl-SNAC as primer. b. Use of diketide-SNAC 5 as primer.