Ketoreduction

Specific CYCs and their interaction with the minimal PKS determine the regiospecificity for the folding and first ring cycUzation of the nascent linear poly- -ketone intermediates. Thus, upon co-action between the minimal PKS and the TcmN or TcmJN CYCs, the nascent linear poly- -ketone intermediates 

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Like the related fatty acid synthases (FASs), polyketide synthases (PKSs) are multifunctional enzymes that catalyze the decarboxylative (Claisen) condensation of simple carboxylic acids, activated as their coenzyme A (CoA) thioesters. While FASs typically use acetyl-CoA as the starter unit and malonyl-CoA as the extender unit, PKSs often employ acetyl- or propionyl-CoA to initiate biosynthesis, and malonyl-, methylmalonyl-, and occasionally ethylmalonyl-CoA or pro-pylmalonyl-CoA as a source of chain-extension units. After each condensation, FASs catalyze the full reduction of the P-ketothioester to a methylene by way of ketoreduction, dehydration, and enoyl reduction (Fig. 3). In contrast, PKSs shortcut the FAS pathway in one of two ways (Fig. 4). The aromatic PKSs (Fig. 4a) leave the P-keto groups substantially intact to produce aromatic products, while the modular PKSs (Fig. 4b) catalyze a variable extent of reduction to yield the so-called complex polyketides. In the latter case, reduction may not occur, or there may be formation of a P-hydroxy, double-bond, or fully saturated methylene additionally, the outcome may vary between different cycles of chain extension (Fig. 4b). This inherent variability in keto reduction, the greater variety of... 

The biosynthesis of polyketides is analogous to the formation of long-chain fatty acids catalyzed by the enzyme fatty acid synthase (FAS). These FASs are multi-enzyme complexes that contain numerous enzyme activities. The complexes condense coenzyme A (CoA) thioesters (usually acetyl, propionyl, or malonyl) followed by a ketoreduction, dehydration, and enoylreduction of the [3-keto moiety of the elongated carbon chain to form specific fatty acid products. These subsequent enzyme activities may or may not be present in the biosynthesis of polyketides. 

Several architectural paradigms are known for polyketide and fatty acid synthases. While the bacterial enzymes are composed of several monofunctional polypeptides which are used during each cycle of chain elongation, fatty acid and polyketide synthases in higher organisms are multifunctional proteins with an individual set of active sites dedicated to each cycle of condensation and ketoreduction. Peptide synthetases also exhibit a one-to-one correspondence between the enzyme sequence and the structure of the product. Together, these systems represent a unique mechanism for the synthesis of biopolymers in which the template and the catalyst are the same molecule. 

Fig. 1. The overall catalytic cycle of polyketide synthases. Within this biosynthetic scheme, different polyketide synthases can show variability with regard to the length of the polyketide chain, the choice of monomer incorporated at each step, the degree of reduction of each P-keto group, and the stereochemistry at each chiral center. For example, the dashed arrows illustrate how the degree of -ketoreduction can vary at any given carbonyl...

Decarboxylative condensation of the malonyl-ACP onto the -ketosynthase-bound growing acyl chain is likely to be analogous to the corresponding reaction catalyzed by the E. coli -ketoacyl synthase. Once formed, the acetoacetyl derivative remains attached to the phosphopantetheine cofactor during subsequent steps of ketoreduction, dehydration, and enoyl reduction, before the growing fatty acid is transferred to the Cys-1305 thiol in preparation for another round of elongation. 

Ketoreduction Ketoreduction requires a KR. The act KR (the only one studied so far) can catalyze reduction of the C-9 carbonyl (counting from the carboxyl end) of any length nascent polyketide backbone studied so far [92, 93,102]. Furthermore, the act KR is compatable with all the minimal PKSs mentioned above. Homologous KRs have been identified in other PKS clusters. These enzymes may also catalyze ketoreduction at C-9 since all the corresponding natural products undergo this modification. In unusual circumstances, C-7 ketoreductions have also been observed with the act KR [93]. 

Similar stereochemical studies have also been conducted on the orsellinic acid synthase from Penicillium cyclopium, a multisubunit enzyme composed of a 130 kDa protein [129, 130]. The catalytic cycle of this PKS is identical to the 6-MSAS cycle, except that it lacks any ketoreduction or dehydration reactions. Unlike 6-MSAS, enolizations occurring during orsellinic acid biosynthesis are not stereospecific. 

It is surprising that one glycosyl transfer step (establishing the C-glycosidic moiety) occurs prior to several other modification steps, in particular an oxygenation, a ketoreduction and a dehydration (no gene has yet been identified responsible for this step), before final glycosyl transfers complete the molecule 281. Urdamycin A (281) is then further modified with amino acids into the dark, discolored urdamycins C, D, E and H [3]. 

Figure 31 Bacillaene biosynthesis, (a) Structure of dihydrobacillaene and bacillaene. PksJ oxidizes the C14 -C15 bond after dihydrobacillaene has been synthesized. Also, note the a-hydroxyacyl N-cap. This particular N-capping has been reported very rarely. (b)a- and /3-Ketoreduction of a-KICto a-HIC. The KR domain of the first PKS module in PksJ is capable of reducing both the a-KIC amide and the /3-ketone in an NAD(P)H-dependent fashion. The order in which these two reductions occur is unknown. Ultimately, keto-reduction is followed by dehydration and enoyl reduction, (c) Theoretical structure of PPant ejection ions used to analyze PksJ ketoreduction. Right PPant ejection ion resulting from IRMPD of Acac-S-PksJ(T3-T4) incubated with PksJ. Mass shift of +2.017 Da corresponds with reduction of the /3-ketone. Left PPant ejection ion resulting from IRMPD of a-KIC-GABA-S-PksJ(T3-T4) incubated with PksJ. Shift of +2.015 Da is observed in PPant ejection ions.

Despite their enormous structural diversity, polyketide metabolites are related by their common derivation from highly functionalised carbon chains whose assemblies are controlled by multifunctional enzyme complexes, the polyketide synthases (PKSs) which, like the closely related fatty acid synthases, catalyse repetitious sequences of decarboxylative condensation reactions between simple acyl thioesters and malonate, as shown in Fig. 3 [7]. Each condensation is followed by a cycle of modifying reactions ketoreduction, dehydration and enoyl reduction. In contrast to fatty acid biosynthesis where the full cycle of essentially reductive modifications normally follow each condensation reduction, the PKSs can use this sequence in a highly selective and controlled manner to assemble polyketide intermediates with an enormous number of permutations of functionality along the chain. As shown in Fig. 3, the reduction sequence can be largely or entirely omitted to produce the classical polyketide intermediate which bears a carbonyl on every alternate carbon and which normally cyclises to aromatic polyketide metabolites. On the other hand, the reductive sequence can be used fully or partially after each condensation to produce highly functionalised intermediates such as the Reduced polyketide in Fig. 3. Basic questions to be answered are (i) what is the actual polyketide intermediate... 

Fig. 3. The assembly of fatty acids, polyketides and reduced polyketides. The reduced polyketide intermediate would be formed from an acetate starter by five successive condensation cycles. The first two cycles are condensations and are followed by condensation-ketoreduction, condensation-ketoreduction-elimination, and finally a full condensation-keto-reduction-elimination-enoyl reduction cycle. Thus the overall reaction sequence is A, A, AB, ABC,ABCD...

Our own studies on the biosynthesis of monocerin (50), Scheme 16, in Dreschlera ravenelii illustrate the problems which commonly arise in this type of study. Incorporations of "H and O-labelled acetates and, gas and analysis by C and "H NMR showed inter alia that the oxygen atoms attached to C-9 and C-11 were acetate derived, so that successive ketoreductions occur with opposite stereochemistry that both hydrogens at C-10 are acetate derived, consistent with ketoreduction occurring during chain assembly and that the extra oxygen at C-4 is derived aerobically [45]. These results are consistent with... 

Scheme 18 illustrates the proposed stages in 6-MSA biosynthesis in which the first and second condensation steps proceed with inversion to give the triketide (63). Ketoreduction gives the alcohol (64) and then elimination followed by a final malonyl condensation generates the tetraketide (65) which cyclises via an intramolecular condensation and enolises to give the aromatic nucleus of (66). In the first set of experiments (J )- and (S)-[l- C, H]nialonales were incubated separately with 6-MSA synthase purified from Penicillium patulum [56]. Isotope incorporations were determined by mass spectrometry. All the possible isotope patterns for retention or loss of the pro-J or pro-S hydrogens from C-3 and C-5 were permutated. Comparison with the actual spectra obtained demonstrated that opposite prochiral hydrogens were eliminated. The absolute stereochemistry was established in an analogous experiment [57] where the chiral malonates were incubated with acetoacetyl CoA rather than acetyl CoA. Subsequent mass spectral analysis showed that it is the Hr proton that is retained at C-3 of 6-MSA and so it can be deduced that the hydrogen at C-5 must be derived from the opposite prochiral hydrogen, Hg. The overall result is summarised in Scheme 18. In a recent collaborative study we have synthesised the triketide alcohol (64) as its NAC thioester and shown that it is indeed a precursor as, on incubation with 6-MSA synthase and malonyl CoA, 6-MSA production is observed [unpublished results]. Current work is aimed at synthesis of both enantiomers of (64) to study the overall stereochemistry of the ketoreduction and elimination reactions.

In their simplest form, polyketides are natural compounds containing alternating carbonyl and methylene groups ( p-polyketones ). The biosynthesis of polyketides begins with the condensation of a starter unit (typically, acetyl-CoA or propionyl-CoA) with an extender unit (commonly malonyl-CoA or methylmalonyl-CoA, followed by decarboxylation of the extender unit (/, 2) (Fig. 1). Repetitive decarboxylative condensations result in lengthening of the polyketide carbon chain, and additional modifications such as ketoreduction, dehydratation, and enoylreduction may also occur (discussed below). 

Figure 3. Relationship between polyketide and fatty acid biosynthesis. The simplest ( minimaV) PKSs possess ketosynthase activity and produce linear polyketide products. In contrast, FASs also catalyze successive ketoreduction-dehydration-enoyl reduction reactions following each condensation. Diverse PKSs may perform none, part, or all of this reductive sequence. KS, ketosynthase KR, ketoreductase DH, dehydratase ER, enoyl reductase.

First ring cyclization must occur before ketoreduction to satisfy the C9 regiospecificity. 

An important question for aromatic polyketide biosynthesis is the timing of the first ring cyclization, relative to ketoreduction. We found that the timing... 

Figure 7. (A) The front side docking results in the R-stereomer during ketoreduction. (B) The back side docking results in the S isomer.

The structures of KS/CLF, KR and ARO/CYC have provided strong clues to the molecular features that result in the observed chain length, ketoreduction and cyclization specificities during polyketide biosynthesis. Based on structural information, the polyketide chain length has been altered by mutations of the CLF residues at the KS/CLF dimer interface. In the future, it should be possible to mutate residues of KS/CLF, KR and ARO/CYC to change the specificity of ketoreduction and cyclization. Therefore, the crystal structures of PKS domains will serve as the blueprints to guide the combinatorial efforts of polyketide biosynthesis. 

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