Ho

co2h

TcmO

co2h

TcmO

Fig. 6. In vitro synthesis of actinorhodin biosynthetic intermediates and its shunt metabolites from acetyl CoA and malonyl CoA by the ActlORFI,2,3 polyketide synthase complex, the Actlll ketoreductase, the ActVII aromatase, ActIV cyclase, and the TcmO methyltransferase malonyl CoA, and NADPH indeed resulted in the formation of 3,8-dihydroxy-1-methyl anthraquinone-2-carboxylic acid (DMAC, 38), the major in vivo product of the complete Act PKS [101]. The identity of 38 was further authenticated by its enzymatic conversion to 8-methoxy DMAC (39), catalyzed by the TcmO methyltransferase in the presence of S-adenosyl methionine (Fig. 6) [231].

With this cell-free assay for the Act PKS activity, Carreras and Khosla attempted to purify the KSa and KSb directly from crude extracts of S. ceolicolor CH999, overexpressing actI-ORF1,2,3, actIII, actIV, and actVII [184]. They soon realized that the purification was complicated by the requirement of several proteins for the PKS activity and that separation of these proteins resulted in loss of activity. While a 40% enriched preparation of KSa and KSb was active in catalyzing polyketide biosynthesis when supplied with purified holo-ACPs, derived from the fren, gra, oct, or tcm PKS gene clusters, and malonyl CoA, a near homogeneous mixture of KSa and KSb was inactive under the same assay conditions. KSa and KS^ were co-purified as an equimolar mixture, according to SDS-PAGE analysis, and existed as an [email protected] heterotetramer in solution based on gel filtration data [184]. However, the purified KSa, KSfb, and holo-ACPs can be activated by addition of a crude extract from S. coelicolor CH999 host strain that lacks genes encoding these proteins and has no PKS activity of its own. The latter verified the integrity of the purified KSa, KSb, and holo-ACPs, and suggested an additional protein essential for the PKS activity. By assaying its ability to complement the purified KSa, KSb, and holo-ACPs in polyketide synthesis, they purified this protein, identified it by N-terminal sequencing as the S. coelicolor fabD MAT, and established that KSa, KSb, holo-ACP, and MAT reconstituted the Act PKS in vitro, synthesizing polyketides from malonyl CoA [184]. Very recently, Simpson and co-workers similarly reconstituted the Act PKS in vitro from individually purified KSa/KSb and ACP with or without MTA, although their goal was mainly to reexamine if MTA is essential in a reconstituted PKS [173]. As discussed in Sect. 3.1.3, these researchers demonstrated that MTA is not required by the minimal Act PKS for polyketide synthesis in vitro as long as the holo-ACP is in large molar excess over KSa/KSb and proposed an alternative mechanism of self-malonyla-tion for loading the malonyl group to the ACP of the PKS complex [173].

Reconstitution of the Act PKS [173, 184] and Tcm PKS [183] in vitro from individually purified KSa, KSb, holo-ACP, and MAT was one of the most exciting advances in the studies of aromatic polyketide biosynthesis, providing excellent model systems for mechanistic studies of bacterial aromatic type II PKS. The methods for Act PKS and Tcm PKS should be applicable to the purification and reconstitution of other aromatic type II PKSs. It will be extremely interesting to examine the effect on polyketide synthesis by swapping functional components of various PKSs in vitro. Summarized here are the highlights that bear fruit directly from the in vitro systems, revealing mechanistic features otherwise inaccessible by in vivo studies.

1. The minimal PKS consists of KSa, KSb, and holo-ACP. Posttranslational phosphopantetheinylation of apo-ACP into holo-ACP is catalyzed by the endogenous ACPS. When holo-ACP is present in limiting concentration, the minimal PKS requires MAT to synthesize polyketide from malonyl CoA. The

Pks Biosynthesis
Fig. 7. Stepwise mechanism for type II PKS catalyzed biosynthesis of aromatic polyketides from malonyl CoA

MAT catalyzes the transfer of the malonyl group from malonyl CoA to the holo-ACP to form malonyl-ACP (40), involving a transient malonyl-MAT species (41) (Fig. 7, path A). ACPS and MAT, both of which are encoded by the fatty acid biosynthetic machinery of the host, provide the functional connections between fatty acid and polyketide biosynthesis.

2. When holo-ACP is present in excess, the minimal PKS is sufficient to support polyketide biosynthesis from malonyl CoA at least in vitro. The loading of malonyl group from malonyl CoA to holo-ACP is a result of self-malonylation of the holo-ACP (Fig. 7, path B), a property unique to the type II PKS ACP.

3. Acetyl CoA is not required for the synthesis of aromatic polyketides with acetate as an starter unit. Instead, the acetate starter is derived from malonyl CoA by decarboxylation of 40 to acetyl-ACP, which is transferred to KSa to initiate polyketide biosynthesis (Fig. 7). Both of these reactions as well as the transfer of the growing poly-b-ketone intermediates between acyl-ACP (42) to acyl-KSa (43) are thought to be catalyzed by the KSa/KSb pair of the PKS. In fact, Carreras and Khosla elegantly demonstrated 41,40,and 43 as covalent intermediates for the Act PKS by malonyl group en route from CoA to polyketide using [14C]malonyl CoA, providing direct evidence for such a mechanism.

4. The full-length poly-b-ketone intermediate, which is tethered to the PKS complex in a thioester linkage at ACP, could spontaneously undergo aberrant cyclization in the absence of additional auxiliary enzymes. Interaction between the PKS complex with additional auxiliary enzymes such as ARO/CYCs dictates subsequent reactions to convert the initial linear poly-b-ketone intermediate into specific aromatic polyketides (Fig. 7).

5. Structural information of the PKSs is emerging. While the stoichiometry of a PKS has yet to be established, and whether or not the difference of the KSa/KSb complex, being a2b2 for the Act PKS and ab for the Tcm PKS, has any particular significance in polyketide synthesis has yet to be determined, it is clear that KSa and KSb can be co-purified and form a tight complex. The latter in turn sequesters holo-ACP to form a ternary PKS complex. This model is consistent with the observation that KSa and KSb together control the chain length of the polyketide product and a heterologous pair of KSa and KSb is often nonfunctional, presumably due to poor interaction between the two subunits or between the KSa/KSb pair and holo-ACP.

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