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Streptomyces coelicolor produces actinorhodin (Act), an aromatic polyketide whose biosynthetic pathway is an important model for the study of type II polyketide synthase (PKS) systems (6). A key component in the Act PKS is a -ketoacyl-acyl carrier protein (ACP) synthase (KS) which presumably forms a heterodimer with a similar protein, KS (previously referred to as chain length factor [14] and recently renamed chain initiation factor [1]). In conjunction with ACP, Act KSKS catalyzes sequential condensation of an acetyl-coenzyme A (CoA) starter unit and seven malonyl-CoA extender units to form an octaketide carbon chain. Although a growing number of studies have demonstrated the ability to manipulate Act PKS and other type II PKSs to generate new compounds (8, 10), relatively little is known about the structural framework for catalysis in these multienzyme systems. Here we report a three-dimensional structure model of Act KS based on the recently resolved X-ray crystal structure of Escherichia coli fatty acid synthase (FAS) -ketoacyl synthase II (KAS II) (7) and its assessment by site-directed mutagenesis.

Structural modeling of Act KS. The condensation reactions catalyzed by KS are conceptually the same as the chain elongation steps of fatty acid biosynthesis carried out by the condensing enzymes of bacterial FASs (15) (Fig. 1A). The crystal structure of E. coli FAS KAS II has recently been solved (7). This enzyme catalyzes the condensation reaction that leads to the elongation of palmitoleic acid (C16:1) to cis-vaccenic acid (C18:1) in fatty acid biosynthesis. Sequence alignment showed that Act KS and E. coli KAS II share 40% identity and 50% similarity (Fig. 1B). Assuming that the same enzymatic mechanism operates for these two proteins, this resemblance in amino acid sequence indicates that they share a similar protein folding pattern and a similar catalytic active site architecture. A three-dimensional model of Act KS was thus generated using the comparative protein modeling server SWISS-MODEL (5), based upon the coordinates of E. coli KAS II (accession code 1kas in the Protein Data Bank, Brookhaven National Laboratory, Upton, N.Y.) (see Fig. 3). The quality of the model has been assessed by using the 3D-1D profile verification method (13) and Prosa II (16), originally built within SWISS-MODEL, as well as by using Procheck (12). The results showed that there are no unfavorable contacts between atoms in this model and that the stereochemical quality of this model is comparable to that of the template structure of E. coli KAS II (data not shown), indicating that Act KS can fold like E. coli KAS II.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   (A) Scheme showing the condensation reaction catalyzed by -ketoacyl-ACP synthase. Steps are labeled as follows: 1, transfer of ACP-bound acyl group to the substrate-binding cysteine residue in KS results in a thioester; 2, a carbanion is generated through decarboxylation of the ACP-bound malonyl group; and 3, nucleophilic attack of this carbanion at the carbonyl carbon atom on the KS-bound thioester results in formation of a carbon-carbon bond. (B) Sequence alignment of Act KS and E. coli FAS KAS II used to build the model of Act KS. Asterisks and dots appear below identical and similar amino acid residues, respectively. Active site Cys residues and the five conserved residues are also highlighted.

Organization of the active site in the Act KS model. Previous studies have already established that a universally conserved Cys residue in various -ketoacyl synthases is the active site nucleophile where the nascent acyl chain is covalently linked (Fig. 1A) (4, 11, 17). In addition, there are five universally conserved residues that are presumably involved in catalysis of the condensation reaction or maintenance of a functional active site configuration (15). In the final energy-minimized Act KS structural model (see Fig. 3), the relative organization of the active site Cys169 residue and the five conserved polar amino acids (His309, His346, Lys341, Asp317, and Glu320) is very similar to that of E. coli KAS II, as analyzed by Swiss-PdbViewer (5). In summary, two His residues (His309 and His346) are in closest proximity to the active site Cys169, with the distances between the N atoms and the S atoms being 4.5 and 3.2 Å, respectively. Lys341 is situated between the two His residues and is within the hydrogen bond distance to the backbone carbonyl oxygen of His309, but the most energy-favorable position of its amino group is pointing away from the S atom of Cys169 (distance, 7.5 Å). Together with Cys169, these three basic residues are located in a solvent-accessible pocket that is lined predominantly by hydrophobic residues. Two acidic residues (Asp317 and Glu320) are located on an  helix near this active site pocket and can be involved in a network of hydrogen bonds that hold a strand containing His309, Gly310, Ser311, Gly312, and Thr313 to form part of the active site pocket.

Functional analysis of the five conserved amino acid residues in Act KS. To assess this model and to examine the importance of the five conserved residues for the activity of Act KS, each of the five conserved residues in Act KS was replaced individually by a neutral amino acid. The changes include His309 to Asn, His346 to Gln, Lys341 to Gln, Asp317 to Asn, and Glu320 to Gln. An E. coli-Streptomyces shuttle vector pRM5 (14), which contains a subset of act biosynthetic genes that specify production of the polyketide aloesaponarin II (Aloe II) and its acidic form, 3,8-hydroxy-1-methyl-anthraquinone-2-carboxylic acid (DMAC), was used as the template for site-directed mutagenesis of actI-orf1 (encoding Act KS) by PCR (9). Replacement of selected amino acid residues was done individually with synthetic oligonucleotide primers, as follows: Asp317 was replaced by Asn (primer 1, 5'-ACCCGGCAGAACAACCGCCACGAGACAGC-3'), Glu320 was replaced by Gln (primer 2, 5'-CAGAACGACCGCCACCAGACAGCGGCGTA-3'), His309 was replaced by Asp (primer 3, 5'-ATCGACTACATCAACGCGAACGGCTCCGG-3'), His346 was replaced by Gln (primer 4, 5'-AACTCGATGGTCGGCCAGTCGCTGGGCGC-3'), and Lys341 was replaced by Gln (primer 5, 5'-TCGATCCAGTCGATGGTCGGCCACTCGCT-3'). A six-His tag was engineered at the C terminus of each protein to facilitate protein purification and provide a unique epitope that distinguished the tagged Act KS from its homologous protein KS. Control experiments demonstrated that fusion of the six-His tag at the C terminus of wild-type Act KS did not affect the function of this protein, as shown by the normal production of polyketide metabolites (data not shown).

View larger version (70K): [in this window] [in a new window]   FIG. 2.   (A) SDS-12% PAGE analysis of total protein extracts from cultures expressing wild-type and mutant Act KS. Lane 1, prestained protein marker (from top to bottom: 43 kDa, 29 kDa, 18.4 kDa, and 14.3 kDa); lane 2, CH999/pDHS3501; lane 3, CH999/pDHS3502; lane 4, CH999/pDHS3503; lane 5, CH999/pDHS3504; lane 6, CH999/pDHS3505; lane 7, CH999/pDHS3401 (wild type); lane 8, CH999. (B) Western blot analysis of the gel used for panel A was carried out with anti-six His antibody. Lanes are as described for panel A. (C) Autoradiography of the SDS-PAGE gel of 3H-cerulenin-labeled Act KS proteins. Lane 1, Act KS mutant (H309N); lane 2, Act KS mutant (H346Q); lane 3, Act KS mutant (K341Q); lane 4, Act KS mutant (E320Q); lane 5, Act KS mutant (D317N); lane 6, wild-type Act KS; lane 7, Ni-NTA column purified protein extracts from CH999.

In vitro labeling of purified Act KS with ³H-cerulenin. Cerulenin, (2S,3R)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide, is a mycotoxin produced by Cephalosporium caerulens that irreversibly inactivates various -ketoacyl-ACP synthases by alkylating the substrate-binding Cys active site residue (4). The reaction between the epoxide group of cerulenin and the nucleophilic thiol of the substrate-binding Cys results in the covalent binding of cerulenin to -ketoacyl synthase (KS). Thus, we used an in vitro ³H-cerulenin labeling assay to address the question of whether any of the mutations introduced into Act KS had affected the reactivity of the Cys169 active site residue. A 1-µg sample of each Ni-nitrilotriacetic acid (NTA) column-purified Act KS protein (the wild-type protein and each mutant protein) was incubated with 1 µCi of ³H-cerulenin at room temperature for 30 min, separated on a sodium dodecyl sulfide (SDS)-12% polyacrylamide gel electrophoresis (PAGE) gel, and then exposed to film. The results demonstrated that each of the mutant Act KS proteins can be labeled by ³H-cerulenin with efficiency equal to that of the native enzyme (Fig. 2C), indicating that none of the mutations affected the formation of the nucleophile in the substrate-binding Cys169 residue. As a negative control, protein extracts from CH999 [after running through Ni-NTA to remove WhiE KS, involved in spore pigment production (2)] revealed no labeling band.

Discussion and conclusion. In the modeled structure of Act KS, the side chain of the active site Cys169 is very close to the side chain of two basic residues: His309 and His346. This organization implies that either one or both of these residues can serve as the base that abstracts a proton from the S atom in the active site residue and thus enhances its nucleophilicity. Accordingly, mutational analysis showed that replacement of either His309 or His346 in Act KS caused a dramatic loss of polyketide production. However, the strains carrying either replacement still produce trace amounts of Aloe II, suggesting that these two residues can at least partially complement each other in function. Furthermore, Act KS bearing the His309Asn or His346Gln mutation can still bind ³H-cerulenin, indicating that formation of the nucleophile in Cys169 has not been disrupted in either mutant. It is thus likely that these two residues can complement each other in aiding the formation of the nucleophile substrate-binding thiol. However, cerulenin is an irreversible inhibitor and the assay can not reveal any potential changes in the cerulenin-binding rate caused by the introduced mutation.

View larger version (63K): [in this window] [in a new window]   FIG. 3.   Ribbon representation of the modeled three-dimensional structure of Act KS. The carbon backbone of the active site Cys169, the five conserved polar residues and Ser347 are in gray, and the side chain oxygen, nitrogen, and sulfur atoms are in red, blue, and yellow, respectively. The figure was prepared using SWISS-PdbViewer 5 and Setor 3.