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Originally published In Press as doi:10.1074/jbc.M703921200 on October 10, 2007

J. Biol. Chem., Vol. 282, Issue 49, 35954-35963, December 7, 2007
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Crystal Structure of the ECH2 Catalytic Domain of CurF from Lyngbya majuscula

INSIGHTS INTO A DECARBOXYLASE INVOLVED IN POLYKETIDE CHAIN β-BRANCHING*Formula

Todd W. Geders{ddagger}§1, Liangcai Gu1, Jonathan C. Mowers{ddagger}2, Haichuan Liu||, William H. Gerwick**, Kristina Håkansson||, David H. Sherman3, and Janet L. Smith{ddagger}4

From the {ddagger}Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, the §Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, the Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, the ||Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, and the **Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093

Received for publication, May 14, 2007 , and in revised form, September 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a β-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH2 domain establishes that the protein is a crotonase superfamily member. Ala78 and Gly118 form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His240 and Lys86, whereas Tyr82 was nonessential. A decarboxylation mechanism is proposed in which His240 serves to stabilize the substrate carboxylate and Lys86 donates a proton to C-4 of the acyl-ACP enolate intermediate to form the {Delta}2 unsaturated isopentenoyl-ACP product. The CurF ECH2 domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr73 may select against CoA-linked substrates by blocking a contact of Arg38 with the CoA adenosine 5'-phosphate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyketides and nonribosomal peptides are important secondary metabolites possessing an array of biological activities and broad chemical diversity (1-4). Type I modular polyketide synthase (PKS)5 and nonribosomal peptide synthetase (NRPS) systems sequentially elongate and modify a growing ketide or peptide chain as it is passed from module to module. The biosynthetic machinery generates tremendous structural variation in these compounds by use of specific reductive domain combinations within modules and/or gene cassettes that encode proteins capable of producing unique functionalities.

The natural product curacin A is a mixed polyketide/nonribosomal peptide produced by the marine cyanobacterium Lyngbya majuscula. Curacin A contains a unique combination of functionalities, including a cyclopropyl ring, a cis-vinyl thiazoline heterocycle, and a terminal alkene (Fig. 1A). Curacin A was first shown in 1994 to possess anti-mitotic activity by the inhibition of tubulin polymerization, cell cycle arrest at the G2/M transition, and anti-proliferative activity against colon, renal, and breast cancer-derived cell lines (5). Further studies have demonstrated rapid, essentially irreversible binding of curacin A to tubulin with potent inhibition of colchicine binding (6).

The biosynthetic gene cluster (cur) for curacin A was recently identified and characterized, revealing that the metabolite is generated by a hybrid type I PKS/NRPS (7). The first six genes in the cur cluster (curA-curF) have been implicated in the formation of the cyclopropyl ring as well as the thiazoline ring of curacin A. Genes within this region include an "HCS cassette" that encodes an acyl carrier protein (ACP) (CurB), a putative ketosynthase (CurC), a 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HCS) (CurD), and two domains containing similarity to enoyl-CoA hydratases (CurE (ECH1) and the N-terminal ~260 residues of CurF (ECH2)) (Fig. 2).


Figure 1
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  		FIGURE 1.
β-Branch introduction by HCS-containing gene cassettes. The position of the β-branch carbon incorporated into the final product of each pathway is indicated by an asterisk. The indeterminate regioisomer of the ECH1 product is shown indicated by a wavy bond. A, pendant methyl group incorporation by CurD/E/F for further tailoring into the cyclopropyl ring of curacin A. B, proposed pathways for methyl (or ethyl) group addition by HCS cassettes. C, proposed pathways for exomethylene group incorporation by HCS cassettes.

 
HCS cassettes are important sources of β-branches in polyketides and allow for the generation of wide chemical diversity at the branch point (Fig. 1). Among the functional groups generated are the cyclopropyl ring of curacin A (7, 8), the vinyl chloride of jamaicamide (9), the exomethylene group of the pederin family of polyketides (10), and the pendant methyl (or ethyl) groups of bacillaene (11, 12), mupirocin (pseudomonic acid A) (13), myxovirescin A (14, 15), and virginiamycin M (16). Subsets of the HCS-containing gene cassette are found in gene clusters that specify production of the bryostatins (17) and leinamycin (18).


Figure 2
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  		FIGURE 2.
Domain organization of CurA-CurF. The CurA-CurF proteins from the curacin A biosynthetic pathway have been implicated in cyclopropyl and thiazoline ring formation. Proteins from an HCS cassette for the introduction of a β-branch in polyketide synthesis include CurB/C/D/E and the ECH2 domain of CurF. Domain labels are: GNATL, loading GCN5-related N-acetyltransferase (52); ACPL, loading acyl carrier protein; KS, β-ketoacyl-ACP synthase; AT, acyl transferase; Hal, halogenase; HCS, 3-hydroxyl-3-methylglutaryl-CoA (HMG-CoA) synthase; ECH, enoyl-CoA hydratase/isomerase; ER, enoyl reductase; DH, β-hydroxy-acyl-ACP dehydratase; Cy, condensaton/cyclization domain; A, adenylation domain; PCP, peptidyl carrier protein.

 
Among PKS gene clusters with HCS cassettes, at least six encode ECH1/ECH2-like enzyme pairs. Recently, the unique catalytic activities of CurE ECH1 and the CurF ECH2 domain have been ascertained (8). CurE ECH1 catalyzes conversion of (S)-HMG-ACP to 3-methylglutaconyl-ACP. Subsequently, the N-terminal ECH2 domain of CurF catalyzes decarboxylation to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A (Fig. 1A). The analogous activities of two other ECH1/ECH2-like enzyme pairs was demonstrated for the HCS cassettes of myxovirescin A and bacillaene (11, 14). Based on the structures of pathway end products, decarboxylation by ECH2-type enzymes are postulated to be followed by proton donation at C-4 leading to a {Delta}2 unsaturated product in curacin A, bacillaene, virginiamycin M, mupirocin, or myxovirescin A or at C-2 leading to a {Delta}3 unsaturated product in jamaicamide and pederin (Fig. 1). Thus, ECH2 represents a critical point of divergence for the generation of correct downstream functionalities in the corresponding natural product. Understanding how these enzymes control product double bond regiochemistry will facilitate the prediction of enzymatic activity for this class of enzymes, as well as aid protein engineering attempts to tailor chemistry for the production of novel natural products.

Enoyl-CoA hydratases belong to the crotonase superfamily, consisting of a wide variety of mechanistically diverse enzymes that exhibit various activities, such as hydratase (19), dehalogenase (20), decarboxylase (21, 22), isomerase (Refs. 22-25; Protein Data Bank code 2F6Q), hydrolase (22), and carbon-carbon bond formation (22) or cleavage (27, 28). Because of this catalytic diversity, the low sequence identity superfamily lacks an invariant catalytic residue, and superfamily membership is established through crystal structures. The common mechanistic theme of the superfamily is the stabilization of an enolate anion intermediate of phosphopantetheine-linked substrates by two backbone amide groups forming an oxyanion hole (29). CurF ECH2 has less than 20% sequence identity to identified members of the superfamily. A unique feature of the ECH2-like enzymes involved in natural product biosynthesis is that they are thought to depend exclusively on ACP-linked substrates in vivo. Structural clues that enable these enzymes to discriminate between CoA and ACP-linked substrates will increase our understanding of the evolutionary changes that have occurred to favor one substrate over another, as well as to control functional group diversity in the final natural product.

In this paper we report the 1.85 Å crystal structure of the wild type, N-terminal ECH2 domain of CurF and the 1.65 Å structure of the corresponding Y82F variant. Modeling of the substrate of CurF ECH2 was performed, and Tyr82, Lys86, and His240 were identified as potential catalytic or substrate-binding residues. Site-directed mutagenesis in a coupled ECH1/ECH2 dehydration/decarboxylation assay with ACP-linked substrates demonstrated that CurF ECH2 His240 and Lys86 are critical to catalysis. Finally, we demonstrated that the 3-methylglutaconyl-ACP substrate is preferred over 3-methylglutaconyl-CoA, a preference that may be controlled by a conserved bulky amino acid residue in the HCS cassette class of decarboxylases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning, Site-directed Mutagenesis, and Protein Expression—The plasmid pMCSG7::CurFd17 was generated by PCR amplification of coding sequence corresponding to residues 17-257 of CurF from the pML9 cosmid DNA (7) and inserted into the vector pMCSG7 (30). The plasmid pMCSG7::CurFd17 was transformed into BL21(DE3) and grown at 37 °C in 2xYT medium to an A600 of 0.6-0.8 in 2L baffle flasks. The cultures were adjusted to 18 °C, and isopropyl β-D-thiogalactopyranoside was added to final concentration of 0.4 mM and allowed to grow 8-16 h with shaking. The cells were harvested by centrifugation, and cell pellets were frozen immediately at -20 °C. Selenomethionine-labeled protein was produced in BL21(DE3) using selenomethionine minimal medium according to the protocol of Guerrero et al. (31). Site-directed mutagenesis was performed by the QuikChange method (Stratagene) (see supplemental materials). All of the constructs were verified by DNA sequencing. CurA (2057-2146)-ACP(II) with a C-terminal His6 tag was constructed by inserting synthetic DNA (a kind gift from Christopher Calderone and Christopher T. Walsh, Harvard Medical School) into pET29a using NdeI and XhoI restriction sites. Protein overexpression and purification of CurA-ACP(II) were performed as described previously for CurB (8).

Protein Purification—All of the steps were performed at 4 °C. The cell pellet from 1 liter of culture was resuspended in 35 ml of buffer A (20 mM Tris, pH 7.9, 500 mM NaCl, 10% glycerol, 20 mM imidazole). Cellytic Express (200-300 mg) was added prior to lysis by sonication (Sigma-Aldrich). The lysate was cleared by centrifugation at >38,000 x g for 45 min. Supernatant was filtered by a 0.45-µm filter, loaded onto a 5-ml HisTrap (GE Healthcare) nickel-nitrilotriacetic acid resin column, and washed with 10 column volumes of buffer A. The protein was eluted with ~200 mM imidazole by a linear gradient of buffer B (20 mM Tris, pH 7.9, 500 mM NaCl, 10% glycerol, 750 mM imidazole). For His tag removal, the fractions were pooled, and buffer was exchanged with buffer A containing 1 mM dithiothreitol and incubated at 4 °C for 24-72 h with 2% (w/w) His-tagged tobacco etch virus protease. The reaction mixture was reloaded onto the HisTrap column, and flow-through fractions were pooled, concentrated, and loaded onto a HiLoad 16/60 Superdex 75 (GE Healthcare) column equilibrated with 20 mM Tris, pH 7.9, 500 mM NaCl, and 10% glycerol. The fractions were then combined, concentrated to 20 mg/ml, and either flash frozen in liquid N2 or stored at 4 °C.

Crystallization—The crystals were grown in 24-72 h at 4 °C by microseeding in hanging drops using the vapor diffusion method. Equal volumes were mixed of protein solution and mother liquor containing 1.3-1.8 M sodium malonate, pH 7.0, 50 mM HEPES, pH 6.8, and 0-25 mM 2-ethanamidoethyl 3-methylbut-2-enethioate. The crystals were harvested in loops and directly frozen by plunging into liquid N2. Both trigonal (P321) and rhombohedral (R32) crystals grew in the above crystallization condition from the same seed stock.

Data Collection and Structure Determination—The diffraction data were collected at 100 K on GM/CA-CAT beamlines 23ID-B and 23ID-D at the Advanced Photon Source in the Argonne National Laboratory (Argonne, IL). The data were processed using the HKL2000 suite (32). Initial phasing was performed using a three-wavelength multiple wavelength anomalous dispersion data set from a single trigonal wild type selenomethionine-labeled protein crystal. SOLVE was used to find the four selenium sites (average figure of merit (FOM) = 0.47, score = 25.07) and for multiple wavelength anomalous dispersion phasing (overall FOM = 0.57) (33). RESOLVE was used for density modification (overall FOM = 0.69) and partial automated model building (34, 35). In both crystal forms, one ECH2 subunit was present in the asymmetric unit. Modeling was completed manually using COOT (36). Refinement was performed using REFMAC5 of the CCP4 suite with TLS (37-39). The refined model was used as a probe structure for molecular replacement using PHASER with data from rhombohedral crystals (40, 41).

Sequence and Structure Analysis—Sequence alignments were performed by T_COFFEE (42). The figures and structure alignments were generated with PyMOL (43). Surface area calculations were calculated using AREAIMOL (44). Identification of structural neighbors was performed by a DALI search (45).

Structure Alignment and Substrate Modeling—C{alpha} atoms of the crotonase core domain of CurF ECH2 (residues 18-222) were aligned with the analogous core region of liganded crotonase superfamily members carboxymethylproline synthase (Ref. 22; Protein Data Bank code 2A81, RMSD = 2.3Å for 167 C{alpha}), methylmalonyl CoA decarboxylase (Ref. 21; Protein Data Bank code 1EF9, RMSD = 1.7Å for 153 C{alpha}), 1,4-dihydroxy-2-naphthoyl-CoA synthase (Ref. 46; Protein Data Bank code 1Q51, RMSD = 1.6Å for 138 C{alpha}), rat enoyl CoA hydratase (Ref. 19; Protein Data Bank code 1DUB [PDB] , RMSD = 1.8Å for 156 C{alpha}), and 4-chlorobenzoyl coenzyme A dehalogenase (Ref. 20; Protein Data Bank code 1NZY [PDB] , RMSD = 2.0Å for 163 C{alpha}) (supplemental Fig. S2). Initial atomic coordinates and topology files for the substrate 3-methylglutaconyl moiety attached to 4-phosphopantetheinic acid were generated using the PRODRG2 server (47). Using the conformation of 4-phosphopantethenic acid bound to rat ECH as a guide (Protein Data Bank code 1DUB [PDB] ), the substrate thioester oxygen was fixed at the position of a water molecule in the oxyanion hole of wild type CurF ECH2. Three water molecules overlapping the modeled substrate were removed. The model was refined by energy minimization using the program CNS (48) in 500 steps of conjugate gradient minimization with no experimental energy terms, no crystallographic symmetry restraints, and fixed main chain positions after the addition of polar hydrogens.

ECH1/ECH2-coupled Enzymatic Assay—The activities of the CurF ECH2 wild type and variants were measured in the ECH1-ECH2 coupled assay, as previously reported (8). In brief, 50 µM (R,S)-HMG-CurA-ACP(II) was incubated with 2 µM ECH1 and ECH2 (wild type or variants) in 50 mM Tris-HCl, pH 7.5, at 37 °C for 1 h. The reactions were terminated by 10% formic acid immediately before loading the reaction mixture on the Jupiter C4 (5 µ, 300 A) reverse phase column (Phenomenex), and the proteins were eluted with CH3CN (0.05% HCOOH and 0.05% CF3COOH)/H2O (0.05% HCOOH and 0.05%CF3COOH). The chromatogram peaks were normalized by 32 Karat software (Beckman Coulter) and subjected to base-line subtraction before peak area calculations.

Preparation of 3-Methylglutaconyl-CoA and ACP—To separate the dehydration and decarboxylation steps catalyzed by ECH1 and ECH2, 3-methylglutaconyl-CoA was prepared by enzymatic dehydration of (R,S)-HMG-CoA. (R,S)-HMG-CoA was incubated with ECH1 at 37 °C for 5 h, and the dehydration product was isolated using XBridge Prep C18 column (Waters, 10 x 250 mm, 5 µm) under the similar HPLC conditions reported (8). The fractions were pooled and lyophilized. ~0.5 mg of 3-methylglutaconyl-CoA was generated from 6 mg of (R,S)-HMG-CoA. 3-Methylglutaconyl-ACP was prepared with the Sfp protocol (8).

ECH1 and ECH2 Assays for ACP and CoA Substrates—(R,S)-HMG-CoA/ACP and 3-methylglutaconyl-CoA/ACP were employed to test the ECH1 and ECH2 activities. 2 µM ECH1 or ECH2 was incubated with 50 µM ACP or CoA substrates in 50 mM Tris-HCl, pH 7.5, at 37 °C for 1 h. The CoA samples were analyzed by XBridge Prep C18 column (Waters, 4.6 x 250 mm, 5 µm) and eluted with MeOH/H2O (10 mM CH3COONH4), and ACP samples were analyzed by Jupiter C4 column and eluted with CH3CN (0.05% HCOOH and 0.05% CF3COOH)/H2O (0.05% HCOOH and 0.05% CF3COOH).

Mass Spectrometry Analysis—Multiply protonated CurA-ACP(II) with or without treatment with ECH1 and/or ECH2 was generated by electrospray ionization (ESI) at 70 µl/h (Apollo ion source; Bruker Daltonics, Billerica, MA) of a solution containing 2 µM CurA-ACP(II) (55:45 CH3CN:H2O with 0.05% HCOOH and 0.05% CF3COOH). All of the mass spectra were collected with an actively shielded 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with a quadrupole front end (APEX-Q, Bruker Daltonics). Ions produced by ESI were externally accumulated in a hexapole for 1 s, transferred via high voltage ion optics, and captured in an ICR cell by gated trapping. This accumulation sequence was looped six times. The ESI capillary voltage was set to -3.8 kV. Nitrogen drying gas (200-250 °C) was employed to assist desolvation of ESI droplets. All of the data were acquired with XMASS software (version 6.1; Bruker Daltonics) in broadband mode from m/z = 200-2000 with 512,000 data points and summed over 10 scans. The mass spectra were analyzed with MIDAS analysis software (49).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structure Determination—Initial crystal screening with a polypeptide including residues 1-257 of CurF produced crystals that diffracted to only ~3.8 Å, but an N-terminal truncation including residues 17-257 yielded crystals that diffracted to beyond 2 Å. The structure was solved by multiple wavelength anomalous dispersion phasing using selenomethionyl CurF ECH2 (Table 1). The resulting model was refined against the 2.0 Å data set in crystal form I and used to solve the structures by molecular replacement in crystal form II (Table 2).


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  		TABLE 1
Data collection

 


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  		TABLE 2
Refinement statistics

 
Structure of the N-terminal ECH2 Domain of CurF—The CurF ECH2 domain possesses the crotonase core fold (residues 17-209) consisting of a central mostly parallel β sheet flanked by {alpha} helices (Fig. 3A). Roughly perpendicular to the main β sheet (β1, β2, β3, β5, and β7) are short β strands (β4, β6, and β8) that are a hallmark of the crotonase superfamily. Crotonase superfamily members can be classified into one of three structural classes based on the conformation of the helical C terminus (22, 29). The helical C terminus ({alpha}9, {alpha}10, and {alpha}11) of CurF ECH2 has the "self-association" fold seen in at least eight other crotonase superfamily members (Refs. 23-25, 28, and 50; Protein Data Bank code 2F6Q), including the two with biotin-independent decarboxylase activities, methylmalonyl CoA decarboxylase (MMCD) (21) and carboxymethylproline synthase (CarB) (22).

Quaternary Structure of the ECH2 Domain of CurF—The trimer structure fundamental to the crotonase superfamily occurs in both crystal forms of ECH2 (Fig. 3B). Subunit contacts are virtually identical in the two crystal forms. A total of 15% of the solvent-accessible surface area of the monomer is buried in the ECH2 trimer (total buried surface area per monomer = 1050 Å2). The extensive buried surface and the lack of water in the subunit interface together indicate that the trimeric association observed in the crystal structure reflects a true quaternary structure for the protein. However, in solution CurF ECH2 also displayed concentration-dependent dissociation (supplemental Fig. S1), indicating a dynamic equilibrium between trimeric and lower oligomeric states.

Active Site—Despite less than 20% sequence identity with other crotonase superfamily members, strong structural similarity of the CurF ECH2 domain to crotonase superfamily members enabled us to align the structures and to identify critical elements of the active site, including the substrate-binding tunnel, active site oxyanion hole, and active site chamber. Structural alignments with several other crotonase superfamily members (supplemental Fig. S2) clearly indicate that the backbone amides of residues Ala78 and Gly118 form the oxyanion hole. These residues follow conserved Gly77 and Gly117, which have backbone conformations only accessible to glycine. In this manner, the peptide planes of residues 77-78 and 117-118 are oriented so their amides can stabilize the proposed enolate anion intermediate by hydrogen bonding. In the crystal structures, a water molecule occupies the oxyanion hole (Fig. 4A). Attempts to obtain crystal structures of complexes with product analogues were unsuccessful. The substrate of CurF ECH2 was then modeled into the active site (see "Experimental Procedures"). Only three polar side chains (Tyr82, Lys86, and His240) are present within the primarily hydrophobic active site chamber (Fig. 4B).


Figure 3
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  		FIGURE 3.
CurF ECH2 structure. A, stereo diagram of the monomer of the CurF ECH2 domain with the active site chamber indicated by an asterisk. B, trimeric structure of the CurF ECH2 domain with the active site chamber indicated by an asterisk. C, multiple sequence alignment of ECH2-like decarboxylases from gene clusters encoding the biosynthesis of curacin (CurF), bacillaene (PksI, 44% identity), virginiamycin M (VirE, 36% identity), myxovirescin A (TaY, 43% identity), mupirocin (MupK, 39% identity), jamaicamide (JamJ, 59% identity), pederin (PedI, 36% identity), and sequences of the two structurally characterized crotonase superfamily decarboxylases (CarB [17% identity] and MMCD [15% identity]). Secondary structure elements of CurF are shown above the alignment, residues of the oxyanion hole are indicated by triangles below the alignment, invariant residues are red, sites of conservative substitution are blue, and similar residues are green. Every tenth residue is underlined.

 
Decarboxylase Activity of Active Site Variants with an ACP-linked Substrate—Based on the active site structure and substrate modeling, the three polar residues in the active site chamber, Tyr82, Lys86, and His240, were tested by site-directed mutagenesis. Proteins possessing Y82F, K86A, K86Q H240A, or H240Q were produced and activity toward ACP-linked substrates was evaluated in a coupled ECH1/ECH2 assay (8). FTICR-MS confirmed that the ECH1 and ECH2 products were produced from the ACP-linked substrate (Fig. 5C and supplemental Fig. S3) 8). The effect of each amino acid substitution was identical in assays with ACP- and CoA-linked substrate (Ref. 8 and data not shown). Substitution of Tyr82 by Phe led to 2-fold reduction in product formation in comparison to wild type (Fig. 5), indicating that Tyr82 plays little or no role in catalysis. Consistent with this conclusion, the Tyr82 phenyl ring in the 1.65 Å crystal structure of the Y82F variant had a similar position to the Phe82 phenyl ring in the wild type enzyme. On the other hand, product formation was reduced more than 20-fold in the K86A, K86Q, H240A, and H240Q variants compared with wild type. Thus, we conclude that Lys86 and His240 play an important role in substrate binding or catalysis. Although the proteins are stable, the CurF ECH2 K86A, K86Q, H240A, and H240Q variants have not yielded crystals.


Figure 4
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  		FIGURE 4.
Active site of CurF ECH2. A, refined 2Fo - Fc electron density contoured at 1 {sigma} from wild type CurF ECH2 domain. The water molecule present in the oxyanion hole is shown with dashed lines to the backbone amides of Ala78 and Gly118. Residues His240, Lys86, and Tyr82 are shown in the foreground. B, stereo diagram of substrate (gray) modeling results highlighting polar residues within8Åofthe modeled substrate carboxylate group. The loop connecting helices {alpha}2 and {alpha}3 covers the active site.

 


Figure 5
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  		FIGURE 5.
ECH1/ECH2 coupled enzymatic assays for ECH2 wild type and mutants. A, HPLC chromatograms showing the (R,S)-HMG-CurA-ACP(II) and associated species before and after ECH1/ECH2 coupled reactions at 37 °C for 1 h; the peaks correspond to (R,S)-HMG-CurA-ACP(II) (peak 1), 3-methylglutaconyl-CurA-ACP(II) (peak 2), and 3-methylcrotonyl-CurA-ACP(II) (peak 3). B, comparison of the peak areas of 3-methylcrotonyl-CurA-ACP(II) for ECH2 wild type and mutants. The data were subjected to normalization and base-line subtraction before the peak area calculation, and the assays were duplicated. C, FTICR-MS identification of the ACP-linked substrate and products. Only the preferred substrate for ECH1,(S)-HMG-ACP, is shown in the reaction scheme (8).

 
Decarboxylase Preference for ACP-linked Substrates over CoA-linked Substrates—CoA- and ACP-linked substrates for ECH2 were prepared by enzymatic synthesis to evaluate the substrate preference of the decarboxylase without the complication of a coupled ECH1/ECH2 assay. CurF ECH2 had a 20-fold preference for the ACP-linked substrate under our standard assay conditions. CurF ECH2 decarboxylated >70% of 3-methylglutaconyl-ACP but only 3% of 3-methylglutaconyl-CoA (Fig. 6). A preference for ACP-linked substrates has not been reported for any member of the crotonase superfamily. Although it presumably has an ACP-linked substrate in vivo, CurE ECH1 differed from CurF ECH2 and showed no substrate preference by providing similar yields of 3-methylglutaconyl-ACP from (R,S)-HMG-ACP and of 3-methylglutaconyl-CoA from (R,S)-HMG-CoA (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The N-terminal ECH2 domain of CurF possesses the crotonase superfamily fold. As is seen with the two other structurally characterized crotonase superfamily members possessing biotin-independent decarboxylase activity (CarB (22) and MMCD (21)), the C-terminal helical domain packs against the monomer in a self-association fold (23). Thus, the active site chamber is formed exclusively by residues within one monomer of the trimer.


Figure 6
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  		FIGURE 6.
Comparison of ACP and CoA substrate preference of ECH1 and ECH2. A, CoA-dependent activities. HPLC traces were monitored at 254 nm. 50 µM (R,S)-HMG-CoA or 3-methylglutaconyl-CoA was treated with 2 µM ECH1 or ECH2. B, ACP-dependent activities. HPLC traces at 280 nm. 50 µM (R,S)-HMG-ACP or 3-methylglutaconyl-ACP was treated with 2 µM ECH1 or ECH2. Peak 1,(R,S)-HMG; peak 2, 3-methylglutaconyl; peak 3, 3-methylcrotonyl. The yields of CoA (A, peak 2) or ACP (B, peak 2) for the reactions of CoA (A, peak 1)/ACP (B, peak 1) treated with ECH1 are similar (~10-15% conversion of substrate to product), but the yields of CoA (A, peak 3) and 3-ACP (B, peak 3) treated with ECH2 are significantly different (~3% for the CoA substrate and >70% for the ACP substrate).

 


Figure 7
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  		FIGURE 7.
Proposed CurF ECH2 mechanism. His240 stabilizes the substrate carboxylate (A), and Lys86 donates a proton to the decarboxylated intermediate (B) to form the product (C). The oxyanion hole is formed by the backbone amides of Ala78 and Gly118.

 
The diversity of chemical reactions catalyzed by crotonase superfamily members and their highly divergent sequences result in little if any conservation within the active site chamber. Nevertheless, the close structural similarity with other crotonase superfamily members suggests strongly that Ala78 and Gly118 form the oxyanion hole of CurF ECH2 (supplemental Fig. S2). The site-directed mutagenesis results show that Lys86 and His240 play crucial roles in catalysis (Fig. 5). We propose a decarboxylation mechanism in which the oxyanion hole anchors the substrate in the active site and stabilizes the enolate anion, His240 hydrogen bonds with the substrate carboxylate, and Lys86 is the proton donor to the C-4 position of the decarboxylated substrate (Fig. 7). The hydrophobic environment of the active site chamber enhances the reactivity of these groups.

Other than Lys86 and His240, Tyr82 is the only polar side chain within the active site chamber. However, elimination of the phenolic hydroxyl by substitution with Phe had only a 2-fold effect on product formation, indicating that Tyr82 does not play a major role in substrate hydrogen bonding or proton donation. Interestingly, Tyr140 of MMCD was proposed to hydrogen bond with the carboxylate group of methylmalonyl CoA in a mechanism analogous to that outlined above (21). Tyr82 of CurF ECH2 and Tyr140 of MMCD are not in analogous parts of the protein structures. Significantly, Tyr82 is not conserved among sequences of decarboxylases in the crotonase superfamily (Fig. 3C).

His240 is conserved in all decarboxylases encoded by HCS cassettes and also in CarB (Fig. 3C). His240 is in an excellent position to assist catalysis by hydrogen bonding with the substrate carboxylate, perhaps orienting the carboxyl optimally to be a leaving group (Fig. 7A). In liganded CarB, His229 is in an identical position to His240 of CurF ECH2 and also was proposed to function by stabilizing the substrate carboxylate (22). The importance of His240 in catalysis was demonstrated in the H240A and H240Q variants of CurF ECH2, in which catalysis was severely impaired (Fig. 5). His240 of CurF ECH2 is the only invariant polar residue within the active site chamber, and we propose that it serves to stabilize the substrate carboxylate here and in other ECH2-like decarboxylases of HCS cassettes (Fig. 3C). An analogous His does not exist in MMCD. The equivalent helix to {alpha}10 of CurF ECH2, which contains His240, is further from the active site chamber in MMCD and forms extensive trimer contacts with the adjacent monomer.

After decarboxylation, proton donation to the substrate C-4 carbon to form isopentenyl-ACP is necessary for enolate collapse to the {Delta}2 unsaturated product (Fig. 7B). Lys86 is the most likely proton donor in CurF ECH2, given the relatively fixed position of the enolate anion in the oxyanion hole during substrate binding and the hydrophobic nature of the active site chamber. Lys86 resides on helix {alpha}2 and has the necessary flexibility and reach to accommodate proton donation at the C-4 carbon of the substrate (Fig. 4B). His240 is not a candidate proton donor because it appears to be unable to reach the C-4 carbon of the decarboxylated intermediate, as required for subsequent product formation, and also unable to move closer to the C-4 carbon. Its backbone is held in place as part of the long helix {alpha}10, and its side chain is positioned by a hydrogen bond with the carbonyl of Thr147 (Fig. 4A) deep within the active site chamber. In contrast, Lys86 is located on helix {alpha}2, the most mobile part of the CurF ECH2 structure as seen in distinctly higher crystallographic temperature factors. This region of the crotonase fold is generally mobile and is completely disordered in some structures.

Proton addition to the C-4 carbon following decarboxylation should produce a {Delta}2 unsaturated product, whereas proton addition to C-2 should yield the {Delta}3 product (Fig. 7). Examples of both reaction routes exist, based on the structures of the PKS products (Fig. 1). Thus, the mechanism of ECH2 is a key determinant of the regiochemistry of the ultimate product of each pathway. The active site chambers of other ECH2-like decarboxylases appear equally as hydrophobic as the CurF ECH2 active site with the exception of the loop between helices {alpha}2 and {alpha}3 (residues 89-95 of CurF; Fig. 3C). This region covers the active site (Fig. 4B) but differs in length and sequence among the ECH2 decarboxylases (Fig. 3C). It is uncertain whether all residues aligned with CurF 89-95 constitute the {alpha}2-{alpha}3 loop in these decarboxylases or whether some of them are part of a longer helix {alpha}3, as in CarB and MMCD (supplemental Fig. S2). Variability in the {alpha}2-{alpha}3 loop is expected among the ECH2 decarboxylases. Each enzyme must possess a unique conformation to accommodate its substrate, often bulkier than the methyl group of the CurF ECH2 substrate (Fig. 1). In addition, a unique proton donor must be positioned to generate specifically a {Delta}2 or {Delta}3 unsaturated product. Candidate proton donors include Lys80, Asp83, or Asp84 of PksI; His68, Asp73, or Asp77 of VirE; Cys74 or Asp75 of TaY; Asp79 or Asp82 of MupK; Glu88, Glu89, Lys92, or Asp95 of JamJ; and Asp2949, Lys2954, Asp2962, or Glu2963 of PedI (Fig. 3C).

The CurF ECH2 domain is the first crotonase superfamily member shown to act preferentially upon ACP-linked substrates. Product formation in vitro with an ACP-linked substrate was 20-fold greater than with a CoA-linked substrate (Fig. 6). Despite the strong preference for ACP-linked substrates by CurF ECH2, structure alignment with other crotonase superfamily enzymes having CoA ligands (supplemental Fig. S2) revealed remarkably few structural changes in the CoA-binding region. Structural motifs that recognize the CoA adenine ring in other crotonase enzymes and the overall backbone conformation that forms the CoA adenosine-binding site are identical in CurF ECH2. One notable difference in CurF ECH2 is the position of the Tyr73 side chain. In the CoA-dependent enzymes, a basic side chain occupies this space and interacts with the 4-phosphate of pantothenic acid and the 5'-phosphate of the CoA nucleotide. CurF Tyr73 is conserved as a bulky phenylalanine or tyrosine residue in the ECH2-like decarboxylases from HCS cassettes with presumed specificity for ACP-linked substrates (Fig. 3C), whereas the analogous position is always a smaller alanine, serine, or valine residue in the CoA-dependent enzymes. Thus, it appears that CoA is a poorer substrate of CurF ECH2 in part because the Tyr73 side chain blocks a basic side chain (Arg38 in CurF) from entering the CoA-binding site. CurF ECH2 containing an alanine substitution at the Tyr73 site was insoluble and has frustrated efforts to test this hypothesis directly. The structural conservation in this region implies a relatively recent evolution of protein function toward ACP-linked substrates.

An intriguing outcome of the CurF ECH2 structure is the apparent symmetry mismatch between its trimeric structure and the fundamentally dimeric structure of several downstream domains in the CurF polypeptide (enoyl reductase, ketosynthase, and dehydratase) (7, 51). The CurF ECH2 domain possesses the crotonase self-association fold with the active site fully formed by each monomer and also demonstrates a capacity for trimer dissociation in solution (supplemental Fig. S1). Thus, the ECH2 domain may be monomeric in the context of full-length CurF, similar to the bacterial fatty acid β-oxidation multienzyme complex in which an N-terminal monomeric crotonase domain is fused to a dimeric dehydrogenase domain (50). Nevertheless, existence of the classical crotonase trimer in the isolated ECH2 domain (Fig. 3B) is strongly suggestive of a trimeric ECH2 within full-length CurF. This could be accomplished in either of two ways. CurF could be a hexamer containing two ECH2 trimers flexibly tethered to three dimers of the downstream domains. Alternatively, CurF could be a dimer in which ECH2 forms a heterotrimer with ECH1 (CurE). Determination of the oligomeric organization of CurF is an on-going investigation.

In summary, the crystal structure of the N-terminal ECH2 domain of CurF PKS-NRPS multifunctional protein from L. majuscula possesses a crotonase fold. The backbone amides of Ala78 and Gly118 form an oxyanion hole. The hydrophobic active site chamber includes only three polar side chains. Of these, Lys86 and His240 are critical for catalytic activity, but Tyr82 is not. Based on assay results and comparisons with other crotonase decarboxylases, His240 is proposed to stabilize the substrate carboxylate, and Lys86 is proposed to donate a proton to the C-4 position of product. These structural features enable CurF ECH2 to generate specifically the {Delta}2 alkene regiochemistry in formation of the key isopentenyl-ACP product during curacin A biosynthesis. CurF ECH2 is highly selective for ACP-linked substrates, whereas CurE ECH1 is nonselective. Strong sequence conservation of both the ECH1 dehydratases (35-95% identity) and the ECH2 decarboxylases (35-60% identity) encoded by HCS cassettes suggests that these domains have similar substrate preferences to the curacin ECH1 and ECH2.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2Q2X, 2Q34, and 2Q35) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by National Institutes of Health Grants DK42303 (to J. L. S.) and GM076477 and the J. G. Searle Professorship (to D. H. S.). This work was also supported in part by a graduate fellowship (to L. G.) from Eli Lilly & Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 These authors contributed equally to this work. Back

2 Supported by the Medical Scientist Training Program at the University of Michigan. Back

3 To whom correspondence may be addressed: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109. Tel.: 734-615-9907; Fax: 734-615-3641; E-mail: davidhs{at}umich.edu.

4 To whom correspondence may be addressed: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109. Tel.: 734-615-9564; Fax: 734-763-6492; E-mail: JanetSmith{at}umich.edu.

5 The abbreviations used are: PKS, polyketide synthase; NRPS, nonribosomal peptide synthetase; CoA, coenzyme A; ECH, enoyl CoA hydratase; HMG, 3-hydroxy-3-methylglutaryl; HCS, HMG-CoA synthase; ACP, acyl carrier protein; RMSD, root mean squared deviation; MMCD, methylmalonyl CoA decarboxylase; HPLC, high performance liquid chromatography; MS, mass spectroscopy; ESI, electrospray ionization; CarB, carboxymethylproline synthase; FTICR, Fourier transform ion cyclotron resonance. Back


   ACKNOWLEDGMENTS
 
We thank David Akey for assistance with domain assignments. We are grateful for the synthetic DNA of CurA-ACP(II) from Christopher Calderone and Christopher T. Walsh. The country of Curaçao and the CARMABI Research Station are acknowledged for access to the source organism, the curacin A-producing L. majuscula. We are indebted to the staff of the GM/CA beamlines (supported by the NIGMS and NCI, National Institutes of Health) at the Advanced Photon Source (supported by the United States Department of Energy).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hill, A. M. (2006) Nat. Prod. Rep. 23, 256-320[CrossRef][Medline] [Order article via Infotrieve]
  2. Staunton, J., and Weissman, K. J. (2001) Nat. Prod. Rep. 18, 380-416[CrossRef][Medline] [Order article via Infotrieve]
  3. Walsh, C. T. (2004) Science 303, 1805-1810[Abstract/Free Full Text]
  4. Fortman, J. L., and Sherman, D. H. (2005) ChemBioChem 6, 960-978[CrossRef][Medline] [Order article via Infotrieve]
  5. Gerwick, W. H., Proteau, P. J., Nagle, D. G., Hamel, E., Blokhin, A., and Slate, D. L. (1994) J. Org. Chem. 59, 1243-1245[CrossRef]
  6. Verdier-Pinard, P., Sitachitta, N., Rossi, J. V., Sackett, D. L., Gerwick, W. H., and Hamel, E. (1999) Arch. Biochem. Biophys. 370, 51-58[CrossRef][Medline] [Order article via Infotrieve]
  7. Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D. H., and Gerwick, W. H. (2004) J. Nat. Prod. 67, 1356-1367[CrossRef][Medline] [Order article via Infotrieve]
  8. Gu, L. C., Jia, J. Y., Liu, H. C., Hakansson, K., Gerwick, W. H., and Sherman, D. H. (2006) J. Am. Chem. Soc. 128, 9014-9015[CrossRef][Medline] [Order article via Infotrieve]
  9. Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M. A., and Gerwick, W. H. (2004) Chem. Biol. 11, 817-833[CrossRef][Medline] [Order article via Infotrieve]
  10. Piel, J., Butzke, D., Fusetani, N., Hui, D., Platzer, M., Wen, G., and Matsunaga, S. (2005) J. Nat. Prod. 68, 472-479[CrossRef][Medline] [Order article via Infotrieve]
  11. Calderone, C. T., Iwig, D. F., Dorrestein, P. C., Kelleher, N. L., and Walsh, C. T. (2007) Chem. Biol. 14, 835-846[CrossRef][Medline] [Order article via Infotrieve]
  12. Butcher, R. A., Schroeder, F. C., Fischbach, M. A., Straight, P. D., Kolter, R., Walsh, C. T., and Clardy, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1506-1509[Abstract/Free Full Text]
  13. El-Sayed, A. K., Hothersall, J., Cooper, S. M., Stephens, E., Simpson, T. J., and Thomas, C. M. (2003) Chem. Biol. 10, 419-430[CrossRef][Medline] [Order article via Infotrieve]
  14. Calderone, C. T., Kowtoniuk, W. E., Kelleher, N. L., Walsh, C. T., and Dorrestein, P. C. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8977-8982[Abstract/Free Full Text]
  15. Simunovic, V., and Muller, R. (2007) ChemBioChem 8, 497-500[CrossRef][Medline] [Order article via Infotrieve]
  16. Pulsawat, N., Kitani, S., and Nihira, T. (2007) Gene (Amst.) 393, 31-42[CrossRef][Medline] [Order article via Infotrieve]
  17. Sudek, S., Lopanik, N. B., Waggoner, L. E., Hildebrand, M., Anderson, C., Liu, H., Patel, A., Sherman, D. H., and Haygood, M. G. (2007) J. Nat. Prod. 70, 67-74[CrossRef][Medline] [Order article via Infotrieve]
  18. Tang, G. L., Cheng, Y. Q., and Shen, B. (2004) Chem. Biol. 11, 33-45[CrossRef][Medline] [Order article via Infotrieve]
  19. Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, 5135-5145[Medline] [Order article via Infotrieve]
  20. Benning, M. M., Taylor, K. L., Liu, R. Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D., and Holden, H. M. (1996) Biochemistry 35, 8103-8109[CrossRef][Medline] [Order article via Infotrieve]
  21. Benning, M. M., Haller, T., Gerlt, J. A., and Holden, H. M. (2000) Biochemistry 39, 4630-4639[CrossRef][Medline] [Order article via Infotrieve]
  22. Sleeman, M. C., Sorensen, J. L., Batchelar, E. T., McDonough, M. A., and Schofield, C. J. (2005) J. Biol. Chem. 280, 34956-34965[Abstract/Free Full Text]
  23. Hubbard, P. A., Yu, W., Schulz, H., and Kim, J. J. (2005) Protein Sci. 14, 1545-1555[Abstract/Free Full Text]
  24. Mursula, A. M., van Aalten, D. M., Hiltunen, J. K., and Wierenga, R. K. (2001) J. Mol. Biol. 309, 845-853[CrossRef][Medline] [Order article via Infotrieve]
  25. Partanen, S. T., Novikov, D. K., Popov, A. N., Mursula, A. M., Hiltunen, J. K., and Wierenga, R. K. (2004) J. Mol. Biol. 342, 1197-1208[CrossRef][Medline] [Order article via Infotrieve]
  26. Lovell, S. C., Davis, I. W., Arendall, W. B., 3rd, de Bakker, P. I., Word, J. M., Prisant, M. G., Richardson, J. S., and Richardson, D. C. (2003) Proteins 50, 437-450[CrossRef][Medline] [Order article via Infotrieve]
  27. Leonard, P. M., Brzozowski, A. M., Lebedev, A., Marshall, C. M., Smith, D. J., Verma, C. S., Walton, N. J., and Grogan, G. (2006) Acta Crystallogr. D Biol. Crystallogr. 62, 1494-1501[CrossRef][Medline] [Order article via Infotrieve]
  28. Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744-1750[Abstract/Free Full Text]
  29. Holden, H. M., Benning, M. M., Haller, T., and Gerlt, J. A. (2001) Acc. Chem. Res. 34, 145-157[CrossRef][Medline] [Order article via Infotrieve]
  30. Stols, L., Gu, M., Dieckman, L., Raffen, R., Collart, F. R., and Donnelly, M. I. (2002) Protein Expression Purif. 25, 8-15[CrossRef][Medline] [Order article via Infotrieve]
  31. Guerrero, S. A., Hecht, H. J., Hofmann, B., Biebl, H., and Singh, M. (2001) Appl. Microbiol. Biotechnol. 56, 718-723[CrossRef][Medline] [Order article via Infotrieve]
  32. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
  33. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
  34. Terwilliger, T. C. (2000) Acta Crystallogr. D Biol. Crystallogr. 56, 965-972[CrossRef][Medline] [Order article via Infotrieve]
  35. Terwilliger, T. C. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 38-44[CrossRef][Medline] [Order article via Infotrieve]
  36. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132[CrossRef][Medline] [Order article via Infotrieve]
  37. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  38. Collaborative Computational Project, N. (1994) Acta Crystallogr. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  39. Painter, J., and Merritt, E. A. (2006) Acta Crystallogr. D Biol. Crystallogr. 62, 439-450[CrossRef][Medline] [Order article via Infotrieve]
  40. Storoni, L. C., McCoy, A. J., and Read, R. J. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 432-438[CrossRef][Medline] [Order article via Infotrieve]
  41. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and Read, R. J. (2005) Acta Crystallogr. D Biol. Crystallogr. 61, 458-464[CrossRef][Medline] [Order article via Infotrieve]
  42. Notredame, C., Higgins, D. G., and Heringa, J. (2000) J. Mol. Biol. 302, 205-217[CrossRef][Medline] [Order article via Infotrieve]
  43. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA
  44. Lee, B., and Richards, F. M. (1971) J. Mol. Biol. 55, 379-400[CrossRef][Medline] [Order article via Infotrieve]
  45. Holm, L., and Sander, C. (1998) Proteins 33, 88-96[CrossRef][Medline] [Order article via Infotrieve]
  46. Truglio, J. J., Theis, K., Feng, Y., Gajda, R., Machutta, C., Tonge, P. J., and Kisker, C. (2003) J. Biol. Chem. 278, 42352-42360[Abstract/Free Full Text]
  47. Schuttelkopf, A. W., and van Aalten, D. M. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 1355-1363[CrossRef][Medline] [Order article via Infotrieve]
  48. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
  49. Senko, M. W., Canterbury, J. D., Guan, S. H., and Marshall, A. G. (1996) Rapid Commun. Mass Spectrom. 10, 1839-1844[CrossRef][Medline] [Order article via Infotrieve]
  50. Ishikawa, M., Tsuchiya, D., Oyama, T., Tsunaka, Y., and Morikawa, K. (2004) EMBO J. 23, 2745-2754[CrossRef][Medline] [Order article via Infotrieve]
  51. Sherman, D. H., and Smith, J. L. (2006) ACS Chem. Biol. 1, 505-509[CrossRef][Medline] [Order article via Infotrieve]
  52. Gu, L., Geders, T. W., Wang, B., Gerwick, W. H., Håkansson, K., Smith, J. L., and Sherman, D. H. (2007) Science. 318, 970-974[Abstract/Free Full Text]

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