The Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. strain SU
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m308198200
ISSN1083-351X
AutoresJames B. Thoden, Zhihao Zhuang, Debra Dunaway‐Mariano, Hazel M. Holden,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoThe 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel β-sheet flanked on one side by a rather long α-helix. The dimers come together to form the tetramer with their α-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their α-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different. The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel β-sheet flanked on one side by a rather long α-helix. The dimers come together to form the tetramer with their α-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their α-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different. During the last century, large quantities of 4-chlorobenzoate or related herbicides and polychlorinated biphenyl pesticides were released into the environment because of commercial production and careless waste disposal (1Cork D.J. Krueger J.P. Adv. App. Microbiol. 1991; 36: 1-66Crossref PubMed Scopus (89) Google Scholar, 2Haggblom M.M. FEMS Microbiol. Rev. 1992; 9: 29-71Crossref PubMed Google Scholar, 3Higson F.K. Adv. Appl. Microbiol. 1992; 37: 135-164Crossref PubMed Scopus (55) Google Scholar, 4Furukawa K. Biodegradation. 1994; 5: 289-300Crossref PubMed Scopus (119) Google Scholar). Strikingly, a variety of soil-dwelling bacteria capable of employing 4-chlorobenzoate as their principal source of carbon have been discovered in richly contaminated areas (5Dunaway-Mariano D. Babbitt P.C. Biodegradation. 1994; 5: 259-276Crossref PubMed Scopus (61) Google Scholar, 6Yi H.-R. Min K.-H. Kim C.-K. Ka J.-O. FEMS Microbiol. Ecol. 2000; 31: 53-60Crossref PubMed Google Scholar, 7Klages U. Lingens F. Hyg. Abt. Orig. 1980; C 1: 215-223Google Scholar). In these microorganisms, 4-chlorobenzoate is first converted to 4-hydroxybenzoate, which is subsequently metabolized via the ortho- or meta-cleavage pathways (5Dunaway-Mariano D. Babbitt P.C. Biodegradation. 1994; 5: 259-276Crossref PubMed Scopus (61) Google Scholar). The 4-chlorobenzoate dehalogenation pathway, as outlined in Scheme 1, consists of three reaction steps catalyzed by 4-chlorobenzoyl-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl-CoA thioesterase (8Scholten J.D. Chang K.-H. Babbitt P.C. Charest H. Sylvestre M. Dunaway-Mariano D. Science. 1991; 253: 182-185Crossref PubMed Scopus (127) Google Scholar). Genes encoding these enzymes are organized within an operon that is under the regulatory control of 4-chlorobenzoate (5Dunaway-Mariano D. Babbitt P.C. Biodegradation. 1994; 5: 259-276Crossref PubMed Scopus (61) Google Scholar). In some bacteria, the gene cluster is located within the chromosomal DNA (12Savard P. Peloquin L. Sylvestre M. J. Bacteriol. 1986; 168: 81-85Crossref PubMed Google Scholar, 13Marks T.S. Wait R. Smith A.R. Quirk A.V. Biochem. Biophys. Res. Commun. 1984; 124: 669-674Crossref PubMed Scopus (35) Google Scholar, 14Chae J.C. Kim Y. Kim Y.C. Zylstra G.J. Kim C.K. Gene (Amst.). 2000; 258: 109-116Crossref PubMed Scopus (30) Google Scholar), whereas in others it is plasmid-encoded (15Layton A.C. Sanseverino J. Wallace W. Corcoran C. Sayler G.S. Appl. Envir. Microbiol. 1992; 58: 399-402Crossref PubMed Google Scholar, 16Ruisinger S. Klages L. Lingens F. Arch. Microbiol. 1976; 110: 253-256Crossref PubMed Scopus (51) Google Scholar).The 4-chlorobenzoyl-CoA pathway operons of certain Arthrobacter and Pseudomonas bacterial strains display significant differences in both gene order and sequences (17Zhuang Z. Gartemann K.-H. Eichenluab R. Dunaway-Mariano D. Appl. Envir. Microbiol. 2003; 69: 2707-2711Crossref PubMed Scopus (30) Google Scholar). At the primary structural level, the amino acid sequence identity between paired Arthrobacter and Pseudomonas ligases is ∼38%, whereas that between paired dehalogenases is ∼50%. Remarkably, however, there is no significant amino acid sequence identity shared between the thioesterases from these species.The three-dimensional structure of the thioesterase from Pseudomonas sp. strain CBS-3 was solved several years ago in this laboratory and was shown to have a "hot dog fold" motif (18Benning M.M. Wesenberg G. Liu R. Taylor K.L. Dunaway-Mariano D. Holden H.M. J. Biol. Chem. 1998; 273: 33572-33579Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 19Thoden J.B. Holden H.M. Zhuang Z. Dunaway-Mariano D. J. Biol. Chem. 2002; 277: 27468-27476Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This type of molecular architecture was first observed in the x-ray structure of β-hydroxydecanoyl thiol ester dehydrase from Escherichia coli (20Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) and has since been found in the (R)-specific enoyl-CoA hydratase from Aeromonas caviae (21Hisano T. Tsuge T. Fukui T. Iwata T. Miki K. Doi Y. J. Biol. Chem. 2003; 278: 617-624Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The structures of these enzymes are dominated by a five-stranded anti-parallel β-sheet that cradles a rather long α-helix of approximately five turns.Here we report a high resolution x-ray crystallographic analysis of the Arthrobacter sp. strain SU 4-hydroxybenzoyl-CoA thioesterase complexed with its products (4-hydroxybenzoate and CoA) or with the substrate analogs, 4-hydroxyphenacyl-CoA or 4-hydroxybenzyl-CoA (Scheme 2). Although the overall topology of the Arthrobacter thioesterase subunit is similar to that described for the Pseudomonas enzyme, its quaternary structure, CoA-binding site, and catalytic platform are different.Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)EXPERIMENTAL PROCEDURESEnzyme Purification and Crystallization—Recombinant Arthrobacter sp. strain SU 4-hydroxybenzoyl-CoA thioesterase (k cat = 6.7 s-1 and K m = 1.2 μm at pH 7.5 and 25 °C; k cat optimal over pH range of 6–10) was purified as previously described (17Zhuang Z. Gartemann K.-H. Eichenluab R. Dunaway-Mariano D. Appl. Envir. Microbiol. 2003; 69: 2707-2711Crossref PubMed Scopus (30) Google Scholar). 4-Hydroxyphenacyl-CoA or 4-hydroxybenzyl-CoA was prepared according to published procedures (19Thoden J.B. Holden H.M. Zhuang Z. Dunaway-Mariano D. J. Biol. Chem. 2002; 277: 27468-27476Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Luo L. Taylor K.L. Xiang H. Wei Y. Zhang W. Dunaway-Mariano D. Biochemistry. 2001; 40: 15684-15692Crossref PubMed Scopus (37) Google Scholar).A search for crystallization conditions was conducted utilizing a sparse matrix screen (designed "in-house") composed of 144 conditions at both room temperature and at 4 °C via the hanging drop method of vapor diffusion. The conditions were tested with both the apo enzyme and that complexed with 1 mm 4-hydroxyphenacyl-CoA. The protein solution, at a concentration of 18 mg/ml, contained 10 mm HEPES (pH 7.5), 150 mm KCl, and 1 mm 1,4-dithio-d,l-threitol. The best crystals were observed growing at room temperature from poly(ethylene glycol) 3400 at pH 7.0 in the presence of 4-hydroxyphenacyl-CoA. Large single crystals were subsequently obtained via hanging drops with precipitant solutions containing 17–20% poly(ethylene glycol) 3400, 100 mm MOPS 1The abbreviation used is: MOPS, 3-(N-morpholino)propanesulfonic acid. (pH 7.0), and 200 mm LiCl. The crystals achieved maximum dimensions of 0.7 mm × 0.7 mm × 0.3 mm in ∼1–2 weeks. They belonged to the trigonal space group P3221 with unit cell dimensions of a = b = 113.2 Å and c = 62.5 Å and contained one dimer in the asymmetric unit. The crystals of the enzyme complexed with either 4-hydroxybenzoyl-CoA or 4-hydroxybenzyl-CoA were prepared in a similar manner. It was never possible to grow crystals of the apo enzyme in a form suitable for a high resolution x-ray analysis.Structural Analysis of the Thioesterase/4-Hydroxyphenacyl-CoA Complex—An initial x-ray data set was collected to 2.6 Å resolution at 4 °C with a Bruker HISTAR area detector system equipped with Supper long mirrors. The x-ray source was CuKα radiation from a Rigaku RU200 x-ray generator operated at 50 kV and 90 mA. The x-ray data were processed with XDS (23Kabsch W. J. Appl. Crystallogr. 1988; 21: 67-71Crossref Scopus (592) Google Scholar, 24Kabsch W. J. Appl. Crystallogr. 1988; 21: 916-924Crossref Scopus (1681) Google Scholar) and internally scaled with XSCALIBRE. 2I. Rayment and G. Wesenberg, unpublished results. The x-ray data collection statistics are presented in Table I.Table IX-ray data collection statisticsData SetResolutionIndependent reflectionsCompletenessRedundancyAvg I/Avg σ(I)R symaR sym = (∑ |I— Ī|∑ I) × 100Å%Enzyme/(4-hydroxyphenacyl-CoA complex)30.0-2.6014,13397.66.86.38.02.72-2.60bStatistics for the highest resolution bin144879.52.31.525.4Mercury derivative30.0-2.6014,40598.65.79.87.52.72-2.60173794.82.91.924.4Enzyme/(4-hydroxyphenacyl-CoA complex)30.0-1.6058,41797.17.435.14.91.66-1.60469381.03.71.739.1Enzyme/(4-hydroxybenzyl-CoA complex)30.0-1.6057,21697.67.239.14.11.66-1.60500786.33.22.129.6Enzyme/(4-hydroxybenzoyl-CoA)30.0-1.9530,62594.13.624.65.12.04-1.95340383.41.94.122.0a R sym = (∑ |I— Ī|∑ I) × 100b Statistics for the highest resolution bin Open table in a new tab One isomorphous heavy atom derivative was prepared by soaking a crystal in 1 mm methylmercury acetate for 1 day. The x-ray data (including Friedel mates) were collected to 2.6 Å. The R-factor between the native and mercury derivative x-ray data sets was 26.8% (where R = Σ|F N - F h|/Σ|F N| × 100, F N is the native structure factor amplitude and F h is the heavy-atom derivative structure factor amplitude). Four heavy atom-binding sites were determined with CNS (25Brunger 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. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 5: 905-921Crossref Scopus (16930) Google Scholar). The positions, occupancies, and temperature factors for these sites were refined with CNS, yielding an overall figure of merit of 0.44 and a phasing power of 1.58. Protein phases were calculated to 2.6 Å resolution with CNS and improved by the method of solvent flipping (as implemented in CNS) to yield a figure of merit of 0.94. Greater than 95% of the residues were built into the solvent flattened electron density map with the graphics software TURBO (26Roussel A. Fontecilla-Camps J.C. Cambillau C. Acta Crystallogr. Sect. A. 1990; 4: 66-67Google Scholar). This "partial" model was subsequently refined via least squares with the program package TnT (27Tronrud D.E. Ten Eyck L.F. Matthews B.W. Acta Crystallogr. Sect. A. 1987; 43: 489-501Crossref Scopus (873) Google Scholar). The resulting electron density maps calculated with coefficients of the form (2F o - F c)or(F o - F c) allowed for the placement of the remainder of the amino acid residues and the 4-hydroxyphenacyl-CoA ligand.High Resolution X-ray Data Collection and Least Squares Refinement—Thioesterase crystals were harvested from hanging drop experiments and equilibrated in a synthetic mother liquor composed of 20% poly(ethylene glycol) 3400, 150 mm KCl, 100 mm LiCl, 1 mm 4-hydroxyphenacyl-CoA, and 100 mm MOPS (pH 7.0). They were then serially transferred to a cryoprotectant solution containing 30% poly(ethylene glycol) 3400, 200 mm KCl, 250 mm LiCl, 15% ethylene glycol, 1 mm 4-hydroxyphenacyl-CoA, and 100 mm MOPS (pH 7.0). The crystals were suspended in a loop of 20-μm nylon and flash frozen in a stream of nitrogen gas. Unit cell dimensions changed to a = b = 112.5 Å and c = 60.6 Å upon cooling to 120 K. A native x-ray data set was collected to 1.6 Å resolution, processed with SAINT (Bruker AXS, Inc.), and scaled as previously described. This structure was solved via molecular replacement with the program AMORE (28Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) employing as the search model the refined structure determined at 2.6 Å resolution. Iterative cycles of least squares refinement and manual model building reduced the R-factor to 18.0% for all measured x-ray data from 30.0 to 1.6 Å resolution. The least squares refinement statistics are presented in Table II.Table IILeast squares refinement statisticsComplex4-Hydroxyphenacyl-CoA4-Hydroxybenzyl-CoAProductsResolution limits (Å)30.0-1.6030.0-1.6030.0-1.95R-factor (overall) (%/no. rflns)aR-factor = (∑|Fo— Fc |∑|Fo |) × 100 where Fo is the observed structure-factor amplitude and Fc is the calculated structure-factor amplitude18.0/5814717.5/5701118.3/30567R-factor (working) (%/no. rflns)17.9/5232017.3/5103418.2/27534R-factor (free) (%/no. rflns)21.9/582721.3/570723.7/3303No. protein atoms2180bThese include multiple conformations for Glu27, Asp39, Val114, Ser128, and Ser140 in Subunit I and Glu27, Asp39, Ser128, and Ser140 in Subunit II2153cThese include multiple conformations for Leu30, Gln94, Lys105, and Ser128 in Subunit I and Glu27, Glu94, and Ser140 in Subunit II2163dThese include multiple conformations for Asp17 in Subunit I and Asp39, Gln94, and Ser120 in Subunit IINo. hetero-atoms428eThese include two 4-hydroxyphenacyl-CoA moieties, four chloride ions, three ethylene glycols, and 296 waters425fThese include two 4-hydroxybenzyl-CoA moieties, one ethylene glycol, and 301 waters357gThese include two CoA molecules, two 4-hydroxybenzoate moieties, and 241 watersBond lengths (Å)0.0120.0120.013Bond angles (deg)2.252.362.24Trigonal planes (Å)0.0070.0060.007General planes (Å)0.0120.0130.012Torsional angles (deg)hThe torsional angles were not restrained during the refinement16.917.217.2a R-factor = (∑|Fo— Fc |∑|Fo |) × 100 where Fo is the observed structure-factor amplitude and Fc is the calculated structure-factor amplitudeb These include multiple conformations for Glu27, Asp39, Val114, Ser128, and Ser140 in Subunit I and Glu27, Asp39, Ser128, and Ser140 in Subunit IIc These include multiple conformations for Leu30, Gln94, Lys105, and Ser128 in Subunit I and Glu27, Glu94, and Ser140 in Subunit IId These include multiple conformations for Asp17 in Subunit I and Asp39, Gln94, and Ser120 in Subunit IIe These include two 4-hydroxyphenacyl-CoA moieties, four chloride ions, three ethylene glycols, and 296 watersf These include two 4-hydroxybenzyl-CoA moieties, one ethylene glycol, and 301 watersg These include two CoA molecules, two 4-hydroxybenzoate moieties, and 241 watersh The torsional angles were not restrained during the refinement Open table in a new tab Structure Determination of the Thioesterase Complexed with 4-Hydroxybenzoyl-CoA (Substrate) or 4-Hydroxybenzyl-CoA—These two structures were solved by difference Fourier techniques with the initial models lacking ordered solvent molecules and ligands. The complex of the enzyme with bound products was prepared by crystallizing the enzyme in the presence of its substrate, 4-hydroxybenzoyl-CoA that was hydrolyzed to 4-hydroxybenzoate (or 4-hydroxybenzoic acid) and CoA. For the sake of simplicity, the ligand will be referred to as 4-hydroxybenzoate, but this does not imply that its protonation state is known. All of the x-ray data for these two complexes were collected in a manner identical to that employed for the thioesterase/4-hydroxyphenacyl-CoA complex as described above. Relevant x-ray data collection and least squares refinement statistics are given in Tables I and II, respectively. In all three complexes, the first 10 and 11 residues were disordered in subunits I and II, respectively. Apart from these disordered N-terminal residues, the electron densities corresponding to both polypeptide chains in the asymmetric unit were continuous throughout the map. The only significant outlier in the Ramachandran plot was Asp39 (in both subunits). This residue is located ∼14 Å from the active site. The dihedral angles adopted by Asp39 produce a bulge in the first β-strand of the sheet. All of the figures were prepared with the software package MOLSCRIPT (29Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).Ligand Binding—The initial velocity of the thioesterase catalyzed hydrolysis of 4-hydroxybenzoyl-CoA was measured by monitoring the decrease in solution absorption at 300 nm resulting from the disappearance of reactant (Δϵ = 11.8 mm-1·cm-1). The reactions were carried out in 50 mm K+-HEPES (pH 7.5, 25 °C) that contained 0.003 μm thioesterase, varying concentrations of 4-hydroxybenzoyl-CoA (1–10 μm) and varying concentrations of 4-hydroxyphenacyl-CoA (0.00925, 0.00185, and 0.037 μm) or 4-hydroxybenzyl-CoA (0.38, 0.76, 1.9, and 3.8 μm). The initial velocity data were analyzed using Equation 1 and the computer program KinetAsyst (IntelliKinetics, PA). V=Vmax[S]/[Km(1+[I]/Ki)+[S]](Eq. 1) where V = initial velocity, V max = maximum velocity, [S] = substrate concentration, K m = Michaelis constant, [I] = inhibitor concentration, and K i = the inhibition constant.RESULTS AND DISCUSSIONInhibitor Binding—The binding constants of the substrate analogs were evaluated by measuring their competitive inhibition constants. Both analogs displayed linear competitive inhibition versus 4-hydroxybenzoyl-CoA. The K i of 4-hydroxybenzyl-CoA was 0.6 ± 0.1 μm, and the K i of 4-hydroxyphenacyl-CoA was 0.003 ± 0.0003 μm. These results indicate that these ligands bind tightly to the substrate-binding site.Overall Structure of the Thioesterase/Product Complex—All of the crystals employed in this investigation contained two subunits/asymmetric unit. For the sake of simplicity, the following discussions will refer only to Subunit II of the x-ray coordinate file unless otherwise indicated. A ribbon representation of the monomer with the bound products, 4-hydroxybenzoate and CoA, is depicted in Fig. 1. The monomer contains 151 amino acid residues, and its topology, referred to as the hot dog fold, is dominated by a six-stranded anti-parallel β-sheet formed by Val37–Met41, Ala46–Val52, Met89–Phe100, His108–Ala118, Trp123–Arg130, and Arg135–Arg149. These β-strands are labeled A–F in Fig. 1. Four of the six β-strands (A, C, D, and F) contain β-bulges at Asp39 (ϕ = -101°, Ψ = -73°), Gln94 (ϕ = -115°, Ψ = -30°), Ile116 (ϕ = -113°, Ψ = -20°), and Cys137 (ϕ = -94°, Ψ = -29°), respectively. In addition to the six β-strands, there are two α-helices delineated by Leu30–Val34 and Gly65–Val84, six Type I turns, and one Type III turn. Nearly 80% of the amino acid residues lie within classical secondary structural elements. The monomer is compact with overall dimensions of ∼38 Å × 49 Å × 35 Å.Fig. 1Ribbon representation of the Arthrobacter thioesterase monomer. The bound ligands, 4-hydroxybenzoate and CoA, are shown in ball-and-stick representations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In that the asymmetric unit contained only two subunits, ultracentrifugation experiments were subsequently performed to define the quaternary structure of the Arthrobacter thioesterase. These experiments were consistent with a tetrameric quaternary structure. The tetramer thus packed in the crystalline lattice with one of its 2-fold rotational axes coincident to a crystallographic dyad. Shown in Fig. 2a is a ribbon representation of the two monomers contained within the asymmetric unit. The tetrameric form of the enzyme, as depicted in Fig. 2b, was generated by rotation of the dimer in the asymmetric unit about the crystallographic 2-fold rotational axis. As can be seen from Fig. 2 (a and b), the quaternary structure of the enzyme can be aptly described as a dimer of dimers with overall dimensions of ∼72 Å × 70 Å × 52 Å.Fig. 2Quaternary structure of the Arthrobacter thioesterase. The quaternary structure of the enzyme can be aptly described as a dimer of dimer. The dimer is shown in panel a with the two separate subunits displayed in blue and green. The 4-hydroxybenzoate and CoA moieties are drawn in ball-and-stick representations with pink- and yellow-filled bonds, respectively. The complete tetramer is depicted in panel b with the long α-helices, one per subunit, displayed as cylinders. Key amino acid residues involved in dimer-dimer interactions are shown in ball-and-stick representations with green-filled bonds. For comparison purposes, the Pseudomonas thioesterase tetramer, complexed with 4-hydroxybenzyl-CoA, is presented in panel c.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The subunit-subunit interface of the dimer is quite extensive with a total buried surface area of ∼3100 Å 2I. Rayment and G. Wesenberg, unpublished results. as calculated according to the method of Lee and Richards (30Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5310) Google Scholar). There are three major regions of subunit-subunit interactions between the two monomers. These intermolecular contacts are formed by Tyr22–Val34 (α-helix), Asp54–Met74 (α-helix), and Gly93–Pro103 (β-strand C) in one subunit and the symmetry-related residues in the second monomer. Indeed, the six-stranded sheets in each monomer come together, through β-strands C, to form a 12-stranded anti-parallel β-sheet in the dimer. The active sites are wedged between the two subunits of the dimer and are separated by ∼24 Å (Fig. 2a). The two major α-helices of the dimer run anti-parallel to one another.The 12-stranded β-sheet of one dimer abuts the β-sheet motif in the second dimer to form the tetramer (Fig. 2b). This packing arrangement is reminiscent to that observed for the dimeric thioesterase II from E. coli (31Li J. Derewenda U. Dauter Z. Smith S. Derewenda Z.S. Nat. Struct. Biol. 2000; 7: 555-559Crossref PubMed Scopus (118) Google Scholar). In this enzyme, each monomer contains two hot dog folds referred to as the "double hot dog." The subunit-subunit interface for the E. coli thioesterase II is likewise built with the β-sheets back to back and the major α-helices facing outwards.Approximately 2125 Å 2I. Rayment and G. Wesenberg, unpublished results. of surface area is buried per monomer upon tetramer formation in the Arthrobacter thioesterase. The dimer-dimer interface is lined with water molecules and various side chains from each subunit including His97, Phe101, His117, and Phe124. As can be seen in Fig. 2b, the histidines at position 97 form a hydrogen bonding ring at the middle of the dimer-dimer interface. The pyrophosphate groups of the CoA moieties project into this interface. Ser120 and Thr121, which lie in a Type I turn defined by Gly119–Thr122, provide hydrogen bonds to the phosphoryl oxygens of the CoA. Specifically, Oγ and the peptidic NH group of Ser120 lie within hydrogen bonding distance to the two α-phosphoryl oxygens, respectively, whereas Oγ and the peptidic NH group of Thr121 are situated within ∼2.5 Å of one of the β-phosphoryl oxygens. Each of the binding sites for CoA is thus formed by three of the four subunits comprising the tetramer.Active Site with Bound Products, 4-Hydroxybenzoate and CoA—As can be seen from Fig. 2a, the 4-hydroxybenzoate is primarily wedged between the two subunits of the dimer pair in the tetramer and specifically between the two major α-helices. A close-up view of the active site is presented in Fig. 3. Key side chains involved in binding the 4-hydroxybenzoate moiety to the protein include Glu73, Thr77, and Glu78 from Subunit II. In addition, the 4-hydroxyl group lies within hydrogen bonding distance to an ordered water molecule, whereas one of the carboxylate oxygens hydrogen bonds to the peptidic NH group of Gly65 in Subunit I. One of the carboxylate oxygens of the 4-hydroxybenzoate ligand is positioned within 2.8 Å of the sulfhydryl group of CoA, indicating that the substrate has indeed been hydrolyzed. The CoA binds to the protein in a quite curved conformation with its ribose adopting the C3′-endo conformation. Only solvent molecules and backbone carbonyl or peptidic NH groups lie within hydrogen bonding distance to the oxygens and nitrogens of the CoA β-mercaptoethylamine and pantothenate units. The pyrophosphate group oxygens of CoA, however, form hydrogen bonds with the side chains of Ser120 and Thr121 situated in the third subunit of the tetramer. Two arginine residues, 102 from Subunit I and 150 from Subunit II, point toward the 3′-phosphate group of the CoA ribose. Additionally, the side chain of Arg150 (Subunit II) runs nearly parallel to the plane of the adenine ring. The amino group at position 6 of the adenine ring forms hydrogen bonds with a water molecule and the carbonyl oxygen of Pro148 from Subunit II.Fig. 3The Arthrobacter thioesterase active site with bound hydrolysis products. Those residues located within ∼3.5 Å of the 4-hydroxybenzoate and CoA molecule are shown. The products are highlighted in yellow-filled bonds. Ordered water molecules are represented by the red spheres. The asterisks indicate those amino acid residues belonging to Subunit II. Ser120 and Thr121, highlighted in pink-filled bonds, belong to Subunit III of the tetramer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structure of the Arthrobacter Thioesterase Complexed with Either 4-Hydroxyphenacyl-CoA or 4-Hydroxybenzyl-CoA—The binding of either 4-hydroxyphenacyl-CoA or 4-hydroxybenzyl-CoA within the thioesterase active site resulted in little perturbation of the polypeptide chain backbone compared with that were observed with bound products. Indeed, the α-carbons for these two complexes both superimpose onto the protein/product complex with a root mean square deviation of 0.17 Å. For all atoms, the three models presented here superimpose upon one another with typical root mean square deviations of 0.45 Å or less. In all three of the complexes, the conformations of the side chains lining the active sites are identical within experimental error. A superposition of the three ligands bound to the protein is presented in Fig. 4.Fig. 4Inhibitor binding to the Arthrobacter thioesterase. A superposition of the two inhibitors, 4-hydroxybenzyl-CoA (pink) and 4-hydroxyphenacyl-CoA (blue) onto the bound hydrolysis products, 4-hydroxybenzoate and CoA (gray).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The K i of 4-hydroxybenzyl-CoA is 0.6 ± 0.1 μm. As can be seen in Scheme 2, the carbonyl functional group of the substrate has been replaced by a methylene bridge in 4-hydroxybenzyl-CoA. The electron density for this inhibitor is indicative of two conformations with the sulfur atoms of the CoA differing by ∼1.8 Å. Interestingly, the K i of 4-hydroxyphenacyl-CoA is considerably lower at 0.003 ± 0.0003 μm. In this inhibitor, an additional methylene group has been i
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