Solution structure and dynamics of oxytetracycline polyketide synthase acyl carrier protein from Streptomyces rimosus

Type II polyketide synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific polyketide product. As part of our ongoing studies into the mechanisms and control of polyketide biosynthesis, we report the second structure of a polyketide synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3-80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the addition of a 13-residue flexible C-terminus. A comparison of the global folds of all structurally characterized ACPs is described, illustrating that PKS ACPs show clear differences as well as similarities to FAS ACPs. (15)N relaxation experiments for the protein backbone also reveal that the long loop between helices I and II is flexible and helix II, a proposed site of protein-protein interactions, shows conformational exchange. The helices of the ACP form a rigid scaffold for the protein, but these are interspersed with an unusual proportion of flexible linker regions.     

Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus

Enediyne antibiotics are known for their potent antitumor activities. One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non-peptide chromophore (1a), and peptide apoprotein. The structurally diverse non-peptide chromophore is responsible for its biological activity. One of its structural components, the naphthoic acid moiety (2,7-dihydroxy-5-methyl-1-naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units. The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6-methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin. Cultures of S. lividans TK24 and S. coelicolor YU105 containing plasmids with NNS were able to produce 2-hydroxy-5-methyl-1-naphthoic acid (2a), a key intermediate of naphthoic acid moiety in NCS. In addition to 2a, a novel product, 2-hydroxy-5-hydroxymethyl-1-naphthoic acid (2d) was isolated. This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds.     

Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase

Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs.     

Tandemly duplicated acyl carrier proteins, which increase polyketide antibiotic production, can apparently function either in parallel or in series

Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged "in series" to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.     

Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site

The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA. During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme. The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S. coelicolorFAS holo-ACP, catalyzed by His(6)-MCAT, gave K(infinity) (M) values of 73 (ACP) and 60 microM (malonyl CoA). A catalytic constant k (infinity) (M) of 450 s(-1) and specificity constants k (infinity) (M)/K (infinity) (M) of 6.2 (ACP) and 7.5 microM(-1) s(-1) (malonyl CoA) were measured. Malonyl transfer to the E. coli FAS holo-ACP, catalyzed by His(6)-MCAT, was less efficient (k (infinity) (M)/K (infinity) (M) was 10% of that of the S. coelicolor ACP). Incubation of MCAT with the serine specific agent PMSF caused inhibition of malonyl transfer to FAS ACPs, and an S97A MCAT mutant was incapable of catalyzing malonyl transfer. Our results show that in the reaction with FAS holo-ACPs the S. coelicolor MCAT is very similar to the E. coli MCAT paradigm in terms of its kinetic mechanism and active site residues. These results indicate that no other active site nucleophile is involved in catalysis as has been suggested to explain recently reported observations.     

Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2–methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2–methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.     

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete beta-ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.     

A three-dimensional model of aromatase cytochrome P450.

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.     

Kinetic analysis of the actinorhodin aromatic polyketide synthase.

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.