Recognition of (2S)-aminomalonyl-acyl carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in zwittermicin A biosynthesis.

Polyketides made by bacterial type I polyketide synthases (PKSs) have a wide range of important biological and pharmacological properties including antibacterial, anticancer, antifungal, antiparasitic, and immunosuppressant activities (1, 2). The diversity in activities of polyketides is due to the vast structural variations afforded by type I PKS enzymology. These enzymes are large, multidomain complexes that systematically condense thioesterified carboxylic acid precursors into a growing polyketide chain. The first of these precursors to be incorporated is known as the starter unit, while the following precursors that elongate the polyketide backbone are known as extender units. The three enzymatic domains required for a single cycle of chain elongation constitute a minimal module: an acyltransferase (AT), an acyl carrier protein (ACP) and a ketosynthase (KS). The AT transfers an acyl moiety from a starter/extender unit to the 4′-phosphopantetheinyl (4′-Ppant) prosthetic group of a holo-ACP, and chain elongation occurs when the KS catalyzes the decarboxylative condensation of two adjacent ACP-linked thioesters. Additional domains such as a ketoreductase (KR), dehydratase (DH), or enoylreductase (ER) may occur, altering the oxidation state of the β-keto group of the unit. Following completion of the elongation cycles, the acyl chain is cleaved from the PKS by a thioesterase domain, releasing the polyketide backbone in either a linear or cyclized form (3). The presence of auxillary domains such as methyltransferases, hydroxylases, halogenases, or glycosyltransferases can result in further modifications to the polyketide (4, 5). In addition to the presence/absence of modifying and β-keto processing domains (KR, DH, ER), the type of starter unit and the number and types of extender units greatly add to the extraordinary structural diversity of the polyketides. While there are a plethora of starter units in polyketide biosynthesis (6), there are only eight extender units currently known (7, 8). These extender units can be categorized into two types. The first class includes those that are covalently linked to coenzyme A (CoA): malonyl-CoA, (2S)-methylmalonyl-CoA, (2S)-ethylmalonyl-CoA, propylmalonyl-CoA, and chloroethylmalonyl-CoA, which incorporate acetyl, propionyl, butyryl, pentanoyl, and chlorobutyryl units, respectively. The second class of extender units includes those that are covalently linked to the 4′-Ppant groups of holo-ACPs: (2R)-methoxymalonyl-ACP, (2S)-aminomalonyl-ACP, and (2R)-hydroxymalonyl-ACP, which elongate a polyketide with methoxyacetyl, glycyl, or glycolyl units, respectively. The first ACP-linked extender unit to be identified, (2R)-methoxymalonyl-ACP occurs in the biosynthesis of several polyketides, including the immunosuppressant FK520 (9), the antitumor antibiotic ansamitocin (10), and the antifungal soraphen A (11). Meanwhile, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP have only been shown to occur in the biosynthesis of the antibiotic zwittermicin A (Figure 1A) (12), although they are proposed as precursors in other biosynthetic pathways (7, 13). Figure 1 Incorporation of two ACP-linked PKS extender units in zwittermicin A biosynthesis. (A) Structure of zwittermicin A with glycyl and glycolyl moieties highlighted. These moieties are derived from the incorporation of (2S)-aminomalonyl-ZmaH and (2R)-hydroxymalonyl-ZmaD, ... Our group is interested in the biosynthesis of zwittermicin A because of its agricultural importance and unusual linear aminopolyol structure (Figure 1A). First isolated from the biocontrol agent Bacillus cereus UW85, zwittermicin A has diverse biological activities including broad-spectrum antibiosis against Bacteria and lower Eukarya, plant disease suppression, and enhancement of the insecticidal activity of Bacillus thuringiensis toxin against lepidopteran larvae (14-16). In addition, zwittermicin A biosynthesis is an excellent model system to study type I PKSs because it utilizes two rare ACP-linked precursors as well as a CoA-linked precursor (malonyl-CoA) and can thus provide insights into both ACP-linked extender unit biosynthesis and AT domain selectivity for ACP-linked versus CoA-linked extender units. Although ACP-linked extender units are not as prevalent, these precursors have generated great interest as potential tools for combinatorial biosynthesis because the substituents on the α-carbons (methoxyl, amino, or hydroxyl) provide hydrogen-bonding potential and functionalities not available through the use of the CoA-linked extender units. The heterologous introduction of methoxyl, amino, or hydroxyl groups to a polyketide may enhance the biological or therapeutic activities of the “unnatural” natural product. Furthermore, the addition of amino and hydroxyl groups may provide reactive chemical handles to facilitate downstream semi-synthetic modifications in the generation of even more structural derivatives. The approach of replacing an AT domain of a pathway with a non-cognate AT domain specific for a different extender unit has been used successfully to generate structural derivatives of important polyketides such as FK520 and the erythromycin precursor 6-deoxyerythronolide B (17, 18). As a first step towards exploring (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP as potential tools for combinatorial biosynthesis, we reconstituted the biosynthetic pathways of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP using heterologously purified enzymes from zwittermicin A biosynthesis (12). In our prior studies of (2S)-aminomalonyl-ACP formation, we have shown that ZmaJ covalently tethers L-serine to the 4′-Ppant group of holo-ZmaH, forming seryl-ZmaH, which is subsequently oxidized by ZmaG (NAD+-dependent) and ZmaI (FAD-dependent) to form (2S)-aminomalonyl-ZmaH (Figure 1C). We also have previously shown that (2R)-hydroxymalonyl-ACP formation occurs in an analogous way: ZmaN covalently tethers 1,3-bisphosphoglycerate (1,3-bPG) to the 4′-Ppant group of holo-ZmaD, forming glyceryl-ZmaD; ZmaG and ZmaE (FAD-dependent) then oxidize this intermediate, forming (2R)-hydroxymalonyl-ZmaD (Figure 1B). The next step towards our goal has been to understand how these extender units are incorporated by the producing organism. Based on the structure of zwittermicin A, we previously proposed that it is made by a hybrid nonribosomal peptide synthetase (NRPS) and PKS megasynthase, with PKS modules utilizing (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP extender units to make the glycyl and glycolyl moieties of the molecule (19). Further characterization of the complete biosynthetic gene cluster has revealed that these modules are encoded by two genes: zmaA, and zmaF (20). ZmaA is a multifunctional enzyme comprised of seven PKS domains and one NRPS component, a condensation (C) domain: KS-KR-ACP-KS-AT-KR-ACP-C (Figure 1B). The first module of ZmaA has been proposed to incorporate (2S)-aminomalonyl-ZmaH while the second module has been proposed to incorporate (2R)-hydroxymalonyl-ZmaD. The presence of KR domains in each of the modules is consistent with the structure of zwittermicin A, which has hydroxyl groups at C9 and C11 of the glycyl and glycolyl moieties. The absence of an AT domain in the first module of ZmaA suggests that the acyltransferase activity is conferred by a trans-acting AT (21); the most likely candidate for providing the function of the “missing” domain is the discrete AT encoded by zmaF. Thus, our proposal is that ZmaF transfers the aminomalonyl moiety from (2S)-aminomalonyl-ZmaH to the first ACP domain of ZmaA (ZmaA-ACP1) (Figure 1C). We have also proposed that the AT domain of ZmaA (ZmaA-AT) transfers the hydroxymalonyl moiety from (2R)-hydroxymalonyl-ZmaD to the second ACP domain of ZmaA (ZmaA-ACP2) (Figure 1D). To test our hypotheses for (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP incorporation, we heterologously overproduced and purified ZmaF and the excised ZmaA domains ZmaA-ACP1, ZmaA-ACP2, and ZmaA-AT and tested them for activity. Here, we report the characterization of ZmaF and ZmaA-AT, ATs specific for (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP, respectively, in zwittermicin A biosynthesis. This work is the first biochemical characterization of ATs specific for ACP-linked PKS extender units and represents an important step towards harnessing the potential of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP to generate novel bioactive molecules through combinatorial biosynthesis.