JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

May 2000, p. 2619–2623 Vol. 182, No. 9

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Structural Modeling and Site-Directed Mutagenesis of theActinorhodin b-Ketoacyl-Acyl Carrier Protein Synthase

MIN HE, MUSTAFA VAROGLU, AND DAVID H. SHERMAN*

Department of Microbiology and Biological Process Technology Institute,University of Minnesota, Minneapolis, Minnesota 55455

Received 10 December 1999/Accepted 9 February 2000

A three-dimensional model of the Streptomyces coelicolor actinorhodin b-ketoacyl synthase (Act KS) wasconstructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensingenzyme b-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. Themodel was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are inclose proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis,while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of thevarious Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formationof the active site nucleophile.

Streptomyces coelicolor produces actinorhodin (Act), an aro-matic polyketide whose biosynthetic pathway is an importantmodel for the study of type II polyketide synthase (PKS) sys-tems (6). A key component in the Act PKS is a b-ketoacyl-acylcarrier protein (ACP) synthase (KSa) which presumably formsa heterodimer with a similar protein, KSb (previously referredto as chain length factor [14] and recently renamed chaininitiation factor [1]). In conjunction with ACP, Act KSaKSb

catalyzes sequential condensation of an acetyl-coenzyme A(CoA) starter unit and seven malonyl-CoA extender units toform an octaketide carbon chain. Although a growing numberof studies have demonstrated the ability to manipulate ActPKS and other type II PKSs to generate new compounds (8,10), relatively little is known about the structural frameworkfor catalysis in these multienzyme systems. Here we report athree-dimensional structure model of Act KSa based on therecently resolved X-ray crystal structure of Escherichia colifatty acid synthase (FAS) b-ketoacyl synthase II (KAS II) (7)and its assessment by site-directed mutagenesis.

Structural modeling of Act KSa. The condensation reactionscatalyzed by KSa are conceptually the same as the chain elon-gation steps of fatty acid biosynthesis carried out by the con-densing enzymes of bacterial FASs (15) (Fig. 1A). The crystalstructure of E. coli FAS KAS II has recently been solved (7).This enzyme catalyzes the condensation reaction that leadsto the elongation of palmitoleic acid (C16:1) to cis-vaccenicacid (C18:1) in fatty acid biosynthesis. Sequence alignmentshowed that Act KSa and E. coli KAS II share 40% identityand 50% similarity (Fig. 1B). Assuming that the same enzy-matic mechanism operates for these two proteins, this resem-blance in amino acid sequence indicates that they share asimilar protein folding pattern and a similar catalytic activesite architecture. A three-dimensional model of Act KSa wasthus generated using the comparative protein modeling server

SWISS-MODEL (5), based upon the coordinates of E. coliKAS 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-1Dprofile verification method (13) and Prosa II (16), originallybuilt within SWISS-MODEL, as well as by using Procheck(12). The results showed that there are no unfavorable contactsbetween atoms in this model and that the stereochemical qual-ity of this model is comparable to that of the template structureof E. coli KAS II (data not shown), indicating that Act KSa canfold like E. coli KAS II.

Organization of the active site in the Act KSa model. Pre-vious studies have already established that a universally con-served Cys residue in various b-ketoacyl synthases is the activesite nucleophile where the nascent acyl chain is covalentlylinked (Fig. 1A) (4, 11, 17). In addition, there are five uni-versally conserved residues that are presumably involved incatalysis of the condensation reaction or maintenance of afunctional active site configuration (15). In the final energy-minimized Act KSa structural model (see Fig. 3), the relativeorganization of the active site Cys169 residue and the fiveconserved polar amino acids (His309, His346, Lys341, Asp317,and Glu320) is very similar to that of E. coli KAS II, as ana-lyzed by Swiss-PdbViewer (5). In summary, two His residues(His309 and His346) are in closest proximity to the active siteCys169, with the distances between the Nε atoms and the Sgatoms being 4.5 and 3.2 Å, respectively. Lys341 is situatedbetween the two His residues and is within the hydrogen bonddistance to the backbone carbonyl oxygen of His309, but themost energy-favorable position of its amino group is pointingaway from the Sg atom of Cys169 (distance, 7.5 Å). Togetherwith Cys169, these three basic residues are located in a solvent-accessible pocket that is lined predominantly by hydrophobicresidues. Two acidic residues (Asp317 and Glu320) are locatedon an a helix near this active site pocket and can be involvedin a network of hydrogen bonds that hold a strand containingHis309, Gly310, Ser311, Gly312, and Thr313 to form part ofthe active site pocket.

* Corresponding author. Mailing address: Department of Microbi-ology, Box 196, 1460 Mayo Memorial Building, 420 Delaware St. S.E.,Minneapolis, MN 55455-0312. Phone: (612) 626-0199. Fax: (612) 624-6641. E-mail: david-s@biosci.umn.edu.

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Functional analysis of the five conserved amino acid resi-dues in Act KSa. To assess this model and to examine theimportance of the five conserved residues for the activity of ActKSa, each of the five conserved residues in Act KSa was re-placed individually by a neutral amino acid. The changes in-clude His309 to Asn, His346 to Gln, Lys341 to Gln, Asp317 toAsn, and Glu320 to Gln. An E. coli-Streptomyces shuttle vector

pRM5 (14), which contains a subset of act biosynthetic genesthat specify production of the polyketide aloesaponarin II(Aloe II) and its acidic form, 3,8-hydroxy-1-methyl-anthraqui-none-2-carboxylic acid (DMAC), was used as the template forsite-directed mutagenesis of actI-orf1 (encoding Act KSa) byPCR (9). Replacement of selected amino acid residues wasdone individually with synthetic oligonucleotide primers, as

FIG. 1. (A) Scheme showing the condensation reaction catalyzed by b-ketoacyl-ACP synthase. Steps are labeled as follows: 1, transfer of ACP-bound acyl groupto 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 alignmentof Act KSa and E. coli FAS KAS II used to build the model of Act KSa. Asterisks and dots appear below identical and similar amino acid residues, respectively. Activesite Cys residues and the five conserved residues are also highlighted.

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follows: Asp317 was replaced by Asn (primer 1, 59-ACCCGGCAGAACAACCGCCACGAGACAGC-39), Glu320 was re-placed by Gln (primer 2, 59-CAGAACGACCGCCACCAGACAGCGGCGTA-39), His309 was replaced by Asp (primer3, 59-ATCGACTACATCAACGCGAACGGCTCCGG-39),His346 was replaced by Gln (primer 4, 59-AACTCGATGGTCGGCCAGTCGCTGGGCGC-39), and Lys341 was replacedby Gln (primer 5, 59-TCGATCCAGTCGATGGTCGGCCACTCGCT-39). A six-His tag was engineered at the C terminus ofeach protein to facilitate protein purification and provide aunique epitope that distinguished the tagged Act KSa from itshomologous protein KSb. Control experiments demonstratedthat fusion of the six-His tag at the C terminus of wild-type ActKSa did not affect the function of this protein, as shown by thenormal production of polyketide metabolites (data not shown).

S. coelicolor CH999 (14), a mutated strain of S. coelicolorwith the entire act gene cluster deleted, was transformed withplasmids bearing each mutation (Table 1). Polyketide produc-

tion in CH999 transformants containing the different con-structs is summarized in Table 2. Briefly, replacement of twoHis residues (His309Asn and His346Gln) greatly impairedthe function of Act KSa, as indicated by the trace amount ofAloe II-DMAC produced by CH999/pDHS3301 (H309N) andCH999/pDHS3302 (H346Q), while replacement of the Lys res-idue (Lys341Gln) and two acidic residues (Asp317Asn andGlu320Gln) resulted in a completely inactive Act KSa, asindicated by the loss of the production of Aloe II-DMAC orany other previously detected polyketide in CH999 contain-ing pDHS3303 (K341Q), pDHS3304 (D317N), or pDHS3305(E320Q). Immunoblotting analysis carried out with equalamounts of protein extracts from each culture with anti-six-Hisantibody demonstrated that each Act KSa mutant protein wasproduced at a similar level (Fig. 2B), indicating that none ofthe mutations introduced had seriously affected the expressionof the mutant Act KSa genes or the stability of the protein. Itis evident that individual replacement of the five conserved

FIG. 2. (A) SDS–12% PAGE analysis of total protein extracts from cultures expressing wild-type and mutant Act KSa. Lane 1, prestained protein marker (fromtop 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-sixHis antibody. Lanes are as described for panel A. (C) Autoradiography of the SDS-PAGE gel of 3H-cerulenin-labeled Act KSa proteins. Lane 1, Act KSa mutant(H309N); lane 2, Act KSa mutant (H346Q); lane 3, Act KSa mutant (K341Q); lane 4, Act KSa mutant (E320Q); lane 5, Act KSa mutant (D317N); lane 6, wild-typeAct KSa; lane 7, Ni-NTA column purified protein extracts from CH999.

TABLE 1. Plasmids and strains used in this study

Plasmid or strain Description Source or reference

PlasmidpRM5 E. coli-Streptomyces shuttle vector containing a subset of act biosynthetic 14pDHS3401 pRM5 derivative encoding wild type Act Act b-KSa with a six-His tag at its C-terminus This workpDHS3501 pDHS3401 derivative carrying gene encoding mutated Act b-KSa (His3093Asn) This workpDHS3502 pDSH3401 derivative carrying gene encoding mutated Act b-KSa (His3463Gln) This workpDHS3503 pDHS3401 derivative carrying gene encoding mutated Act b-KSa (Lys3413Gln) This workpDHS3504 pDHS3401 derivative carrying gene encoding mutanted Act b-KSa (Asp3173Asn) This workpDHS3505 pDHS3401 derivative carrying gene encoding mutated Act b-KSa (Glu3203Gln) This work

StrainS. coelicolor CH999 proA1 argA1 SCP12 SCP22 redE60 Dact 14

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residues has a direct affect on the activity of Act KSa, confirm-ing the functional importance of each that is suggested by themodeled structure.

In vitro labeling of purified Act KSa with 3H-cerulenin. Ceru-lenin, (2S,3R)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide, is amycotoxin produced by Cephalosporium caerulens that irrevers-ibly inactivates various b-ketoacyl-ACP synthases by alkylatingthe substrate-binding Cys active site residue (4). The reactionbetween the epoxide group of cerulenin and the nucleophilicthiol of the substrate-binding Cys results in the covalent bind-ing of cerulenin to b-ketoacyl synthase (KS). Thus, we used anin vitro 3H-cerulenin labeling assay to address the question ofwhether any of the mutations introduced into Act KSa hadaffected the reactivity of the Cys169 active site residue. A 1-mgsample of each Ni-nitrilotriacetic acid (NTA) column-purifiedAct KSa protein (the wild-type protein and each mutant pro-tein) was incubated with 1 mCi of 3H-cerulenin at room tem-perature for 30 min, separated on a sodium dodecyl sulfide

FIG. 3. Ribbon representation of the modeled three-dimensional structure of Act KSa. The carbon backbone of the active site Cys169, the five conserved polarresidues 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 usingSWISS-PdbViewer 5 and Setor 3.

TABLE 2. Polyketide production by S. coelicolor CH9999containing various constructs

Expression system Polyketideproduced

CH999 ............................................................................. —c

CH999/pRM5 .................................................................Aloe II-DMACa

CH999/pDHS3401 .........................................................Aloe II-DMACCH999/pDHS3501(His309-Asn) ..................................Aloe II-DMACb

CH999/pDHS3502(His346-Gln)...................................Aloe II-DMACb

CH999/pDHS3503(Lys341-Gln)................................... —CH999/pDHS3504(Asp317-Asn).................................. —CH999/pDHS3505(Gln320-Gln) .................................. —

a DMAC is the acidic form of Aloe II.b Level of production was less than 1% of the wild-type production level.c No Aloe II-DMAC produced.

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(SDS)–12% polyacrylamide gel electrophoresis (PAGE) gel,and then exposed to film. The results demonstrated that eachof the mutant Act KSa proteins can be labeled by 3H-ceruleninwith efficiency equal to that of the native enzyme (Fig. 2C),indicating that none of the mutations affected the formation ofthe nucleophile in the substrate-binding Cys169 residue. As anegative control, protein extracts from CH999 [after runningthrough Ni-NTA to remove WhiE KSa, involved in spore pig-ment production (2)] revealed no labeling band.

Discussion and conclusion. In the modeled structure of ActKSa, the side chain of the active site Cys169 is very close to theside chain of two basic residues: His309 and His346. Thisorganization implies that either one or both of these residuescan serve as the base that abstracts a proton from the Sg atomin the active site residue and thus enhances its nucleophilicity.Accordingly, mutational analysis showed that replacement ofeither His309 or His346 in Act KSa caused a dramatic loss ofpolyketide production. However, the strains carrying eitherreplacement still produce trace amounts of Aloe II, suggest-ing that these two residues can at least partially complementeach other in function. Furthermore, Act KSa bearing theHis309Asn or His346Gln mutation can still bind 3H-cerulenin,indicating that formation of the nucleophile in Cys169 has notbeen disrupted in either mutant. It is thus likely that these tworesidues can complement each other in aiding the formation ofthe nucleophile substrate-binding thiol. However, cerulenin isan irreversible inhibitor and the assay can not reveal any po-tential changes in the cerulenin-binding rate caused by theintroduced mutation.

Interestingly, the modeled structure suggests that Lys341,Asp317, and Glu320 are not directly involved in the function ofCys169. In agreement with this prediction, none of the replace-ments involving these three residues affected the ability of theactive site Cys169 to react with cerulenin. However, the mod-eled Act KSa structure does indicate that each of the threeresidues can be involved in a hydrogen bond network thatappears to be critical for securing the active site geometry.Modeling of the mutated enzyme indicated that replacementof Lys341, Asp317, and Glu320 with the residues chosen in thisstudy would either disrupt or reduce these hydrogen bonds(data not shown) and thus result in inactive proteins, as sup-ported by the fact that the corresponding strains of S. coelicolorcompletely lost polyketide production. However, the preciseidentification of the roles of these residues to the structuralintegrity of the active site pocket will have to await furtherstructural analysis of the wild-type and the mutant KSa pro-teins.

It is worth noting that a partially conserved Ser347 is alsolocated near the predicted active site pocket in this modeledstructure; however, its side chain is pointing away from Cys169in the active site pocket and does not appear to have anysignificant functional or structural role (Fig. 3). This configu-ration is consistent with previous site-directed mutagenesisstudies, which showed that replacement of Ser347 with Leuresulted in only slightly reduced polyketide production (11).This result, taken together with the studies described above,provides strong support for the modeled active site structure ofAct KSa.

The results presented here have confirmed the functionalimportance of five amino acids that are uniformly conserved in

various b-ketoacyl-ACP synthases, as originally inferred frommultiple sequence alignments of various KSs and the crystalstructure of E. coli KAS II. Our data also strongly support theclaim that b-ketoacyl-ACP synthases from different types ofFASs and PKSs may share a common protein folding patternand a common molecular architecture for catalysis. It is evi-dent that, in the future, the structure of various type II b-ke-toacyl-ACP synthases producing different polyketides could bemodeled in a similar way. Comparison of these structuresmight reveal the subtle structural differences around the sub-strate binding pocket that contribute to the creation of struc-tural diversity in polyketide products and contribute to therational design of enzyme structures for the production ofnovel polyketides.

We thank C. Khosla for providing CH999 and pRM5, M. Siggaard-Andersen for providing 3H-cerulenin, and J. Thompson for assistancewith analyzing the modeled protein structure.

This work was supported by NIH grant GM48562 and a grant fromthe Office of Naval Research. M. V. was the recipient of a PostdoctoralFellowship from the National Cancer Institute (CA09138).

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