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FEBS Letters 585 (2011) 459–464

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Structural basis for carbon dioxide binding by 2-ketopropyl coenzyme M oxidoreductase/carboxylase q ⇑ Arti S. Pandey a, David W. Mulder a, Scott A. Ensign b, John W. Peters a,

a Department of Chemistry and and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, MT 59717, United States b Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, United States

article info abstract

Article history: The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been deter- Received 1 November 2010 mined in a state in which CO2 is observed providing insights into the mechanism of carboxylation. Revised 17 December 2010 In the substrate encapsulated state of the , CO2 is bound at the base of a narrow hydrophobic Accepted 20 December 2010 substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic Available online 27 December 2010 to hydrophilic environment where CO2 is located in position for attack on the carbanion of the keto- Edited by Stuart Ferguson propyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively dis- criminates against H2O and prevents protonation of the ketopropyl leaving group. Keywords: Disulfide oxidoreductase Structured summary: Carboxylase 2-KPCC binds to 2-KPCC by x-ray crystallography (View interaction) Carbon dioxide Acetoacetate Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Coenzyme M

1. Introduction NADP+-dependent carboxylase that reductively cleaves and car- boxylates a thioether substrate. Xanthobacter Py2 is one of several microorganisms that can grow 2-KPCC is a member of the disulfide oxidoreductases (DSOR), a on ethylene, propylene or butylene as sole carbon and energy source. family of which catalyze reductive cleavage of a disulfide- During aerobic of short chain alkenes by Xanthobacter bound substrate followed by subsequent protonation of leaving autotrophicus Py2, coenzyme M is conjugated to highly reactive, groups (for a review, see [5]). At the core of DSOR enzymes there short chain epoxides for subsequent reductive carboxylation to is a highly redox active cysteine pair, separated by 4 residues, at b-keto acids [1–3]. The last step of the pathway is facilitated by which substrate reduction occurs [6]. The reaction catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC), a 2-KPCC is similar to that of other DSORs, but is unique in two flavin adenine dinucleotide (FAD)-containing nicotinamide adenine ways: (i) 2-KPCC cleaves a thioether rather than a disulfide bond, dinucleotide phosphate (NADP)-dependent oxidoreductase. 2-KPCC and (ii) 2-KPCC catalyzes reversible carboxylation. The catalytic catalyzes reversible carboxylation of ketopropyl-CoM thioether mechanism of 2-KPCC was originally proposed to involve a base- (2-K-S-CoM), yielding acetoacetate and regenerating coenzyme M stabilized enolate intermediate of acetone (Scheme 2) [4]. (2-mercaptoethanesulfonate) (CoM-SH) (Scheme 1) [4]. Subsequent structural characterization of 2-K-S-CoM bound en- In contrast to the role of CoM-SH as a C1 carrier in - zyme suggested that the catalytic acid for stabilization of the ace-

esis, CoM-SH acts as a C3 carrier throughout multiple steps in the tone enolate is a histidine bound H2O molecule [7]. In contrast with epoxide carboxylation pathway. 2-KPCC is an FAD-containing other DSOR mechanisms, which require protonation of the leaving group for productive catalysis, protonation would result in dead- end, irreversible production of acetone, which occurs only when

Abbreviations: 2-KPCC, 2-ketopropyl coenzyme M oxidoreductase/carboxylase; CO2 is not present (Scheme 3) [4]. FAD, flavin adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phos- In the presence of CO2, protonation to form acetone is negligi- phate; 2-K-S-CoM, 2-ketopropyl coenzyme M; CoM-SH, coenzyme M (2-mercap- ble, and nearly all 2-K-S-CoM is converted into acetoacetate and toethanesulfonate); DSOR, disulfide oxidoreductase; RMSD, root mean square CoM [4]. deviation; Rubisco, 1,5-bisphosphate carboxylase/oxidase q The reverse of the physiological reaction, decarboxylation of ace- Coordinates and structure factors have been deposited in the Protein Data Bank + under the accession code 3Q6J. toacetate in the presence of CoM-SH and NADP , also presumably ⇑ Corresponding author. Fax: +1 406 994 7212. occurs through the formation of an enolate intermediate of acetone E-mail address: [email protected] (J.W. Peters). and yields 2-K-S-CoM (Scheme 4) [4].

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.12.035 460 A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464

Scheme 3.

Scheme 1.

Scheme 4.

Stabilization of a carbanion through charge delocalization with an adjacent carbonyl or imine that has been polarized by coordina- tion to a Lewis acid is widely used in enzyme-catalyzed decarbox- ylation, isomerization and aldol reactions [8,9]. Other enzymatic carboxylation mechanisms include Schiff-base formation between a lysine and to provide an electron sink [10,11]. This strategy is not used by 2-KPCC, which has an active site devoid of lysine residues. By soaking crystals of FAD-containing 2-KPCC with NADP+ and products CoM-SH and acetoacetate, we have enabled the reverse of the physiological reaction resulting in catalytic decarboxylation

of acetoacetate, producing the substrates 2-K-S-CoM and CO2. The structure of this complex represents the first direct visualization of

the mode of CO2 recognition used by 2-KPCC. Comparison of the CO2 binding site of 2-KPCC with other known enzyme/CO2 com- plexes reveals a hydrophobic channel with unique structural fea-

tures for CO2 insertion and a mode of CO2 recognition that aids Scheme 2. in discrimination against H2O at the active site.

Fig. 1. (A) Cartoon representation of the structure of 2-KPCC observed in a state with bound CO2. The overall structure is colored according to subunit (subunit 1, blue; subunit 2, purple) and a unique loop region is colored orange. The transparent surface rendering (green) depicts the active site formed at the subunit interface. The zoom region displays the active site area as sticks and depicts CO2 bound along with bicarbonate, 2-K-S-CoM and selected neighboring residues with side chains colored according to hydrophobicity/hydrophilicity (brown/cyan). The blue mesh represents the 2Fo À Fc map for CO2, bicarbonate and H2O molecules contoured at 1.3r. Hydrogen bonds at the active site are shown as dashes with distance in angstroms. The residue label color corresponds to the residues contributed by the two subunits. Atomic coloring scheme for

CO2, bicarbonate, and 2-K-S-CoM: C, green; O, red; S, orange. All figures were generated with PyMOL [28]. A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464 461

2. Materials and methods from an H2O molecule that in turn is 2.7 Å away from the CO2 mol- ecule (Fig. 1). In relation to the 2-K-S-CoM molecule, the carbon 2.1. Expression, purification, and crystallization atom of bicarbonate is 2.7 Å away from the carbonyl oxygen of the ketopropyl group and 4.6 Å away from the methyl carbon of Expression of X. autotrophicus Py2 cells [12] and purification of the ketopropyl group. 2-KPCC [13] were carried out as described previously. Crystals The presence of CO2 was observed in one subunit of the struc- were obtained by incubating purified 2-KPCC (30 mg/ml) with ture, while the other subunit shows CoM-SH and ambiguous den- acetoacetate, CoM-SH, and NADP+ added in sequential order for sity that may be consistent with bicarbonate and acetone. Because 10 min prior to addition of an equal amount of precipitating solu- decarboxylation of acetoacetate in the presence of CoM-SH and tion of 0.1 M ammonium acetate, 0.085 M sodium citrate pH 5.6, NADP+ is presumed to occur through the formation of an enolate 25% PEG 4000 and 30% glycerol. The crystals were grown at room intermediate of acetone (Scheme 4), this conformation could rep- temperature using vapor diffusion conditions described previously resent an immediate mechanistic decarboxylation step of acetoac- [14], but reduction with DTT was not performed prior to data etate without concomitant formation of 2-K-S-CoM and capture of collection. CO2. Interestingly, the acetone molecule observed in the second subunit is stabilized by an H2O molecule which is in turn stabilized 2.2. Structure determination and refinement by the carbonyl oxygen of Ala-430, lending support that it could be the enolate intermediate of acetone decarboxylation product from The data were collected from a single cryo-cooled crystal at acetoacetate. It is not uncommon for crystal structures to have dif- SSRL beamline 7–1. The raw data were processed with MOSFLM ferent reaction intermediates in the different subunits. As an [15] and scaled and merged with the CCP4 program suite [16]. example, the crystal structure of a dioxygenase contains three dif- Crystals of 2-KPCC belong to the monoclinic space group P21 with ferent reaction intermediates in the different subunits of the one dimer in the asymmetric unit with the following unit cell homotetrameric enzyme [23]. parameters, a = 88.1 Å, b = 60.0 Å, c = 105.9 Å, and b = 102.4°. The substrate bound crystal structure of 2-KPCC was used as a starting 3.2. Hydrophobic channel for CO2 insertion model (PDB ID: 1MO9), as the crystals are nearly isomorphous. Iterative cycles of refinement and model building using REFMAC The resulting 2-KPCC X-ray crystal structure with 2-K-S-CoM

(version 5.5.0109) [17] and COOT [18], respectively, were per- and CO2 has a hydrophobic channel leading from bulk solvent to formed until convergence, and the structure was refined to the res- the hydrophobic CO2 binding pocket adjacent to the active site olution of 1.92 Å. Data collection, processing, and refinement and the methylene group of the thioether linkage (Fig. 2A). The statistics are given in Supplementary Table 1. Superimposition hydrophobic channel is presumably the pathway for CO2 insertion of the structure on the substrate bound crystal structure of 2-KPCC has an overall root mean square deviation (RMSD) value of 0.21 Å.

3. Results

3.1. The active site of CO2 bound 2-KPCC

Crystals of 2-KPCC soaked with products acetoacetate and CoM-

SH contain linear electron density consistent with a CO2 molecule in a hydrophobic pocket of one of the subunits adjacent to a newly synthesized 2-K-S-CoM molecule (Fig. 1), indicating that enzy- matic decarboxylation of acetoacetate has occurred. Previous work has shown that the substrate 2-K-S-CoM becomes encapsulated within the protein due to ligand-induced conformational changes

[14].CO2 refines well into the linear density with limited residuals and strategically resides at the hydrophobic interface between the two subunits of the enzyme (Fig. 1). The CO2 molecule has very limited water interactions, differing from previously determined

CO2 bound structures where hydrogen bonding for the positioning or polarization of the substrate is not constrained by having to dis- criminate between H2O and CO2 [19–22]. Near the CO2 position, trigonal planar density was observed at a location termed as the alternate anion binding site which was pro- posed earlier to be the site of acetoacetate binding (Fig. 1) [14]. The alternate anion binding site presumably serves to stabilize the neg- ative charge buildup during acetoacetate formation [14]. On the basis of the presence of H2O molecules of which positions are con- served in all forms of 2-KPCC solved structures, present within hydrogen bonding distances to the apical atoms of the trigonal pla- nar density, a bicarbonate molecule was modeled at this site. Ace- Fig. 2. (A) Surface representation of the hydrophobic channel where CO2 (ball and tate from the crystallization buffer was ruled out based on the fact stick representation) enters the enzyme and resides at its base. (B) Proline flanked that this trigonal planar density is not seen in other crystals of 2- loop region (orange) which aids in formation of the CO2 insertion channel. (C and D) KPCC formed with the same crystallization mix. Two of the oxygen Metal binding site at the base of proline flanked loop region. The central atom of the octahedral binding site is depicted in green and shown coordinated (black dashes) atoms of the bicarbonate molecule make hydrogen bonds to two to neighboring residues and H2O molecule (red). The blue mesh (D) represents the

H2O molecules (2.7 and 2.9 Å) and the third oxygen atom is 2.6 Å 2Fo À Fc map contoured at 1.3r and hydrogen bond distance are in angstroms. 462 A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464 prior to carboxylation. Tight closing of a proline flanked loop 4.2. The bicarbonate binding site of 2-KPCC region assists in the formation of the hydrophobic CO2 channel. Featuring two proline residues (Pro-420 and Pro-421) in the cis Both active sites show the presence of trigonal planar density conformation at one of the termini of the region and a proline res- modeled as bicarbonate at the alternate anion binding site pro- idue (Pro-432) at the other terminus, the region is tightly looped posed earlier to be the site of acetoacetate binding [14]. Precedent and structurally unique among DSOR enzymes (Fig. 2B). The region for bicarbonate recognition by the alternate ion binding site ap- is further stabilized near its base of one of the termini by an octa- pears in published structures of b-carbonic anhydrases from Hae- hedral metal binding site from coordination by the carbonyl atoms mophilus influenzae and Escherichia coli, where bicarbonate of Leu-431 (2.5 Å), Val-429 (2.5 Å), Ala-447 (2.4 Å), Leu-427 (3.2 Å), molecules were found at an inhibitory site independent from the

Ser-450 (2.4 Å) and an H2O molecule (2.3 Å) (Fig. 2C and D). enzyme active site [25]. The authors identified two H2O molecules Although the identity of the metal could not be determined unam- bridging the bicarbonate molecule and protein functional groups biguously by ICP-MS, it is thought to be a metal cation such as Mg2+ (PDB ID’s 2ESF & 2A8D). These residues are structurally conserved given the octahedral coordination geometry of the site. in b-carbonic anhydrase structures from Pisum sativum [26] and

4. Discussion

4.1. The binding and recognition mode of CO2

Unlike the four previously determined crystal structures of CO2 bound at enzyme active sites [19–22] there are limited interactions in the form of hydrogen bonds in 2-KPCC that could contribute to polarizing the substrate CO2.CO2 is strategically located at the base of a hydrophobic channel at the subunit interface of the enzyme (Fig. 1 and Fig. 2). This limited polarization is mechanistically sig- nificant in maintaining the electrophilic character of the carbon atom of CO2. There is a single hydrogen bond between an oxygen atom of CO2 and an H2O molecule bridged to an adjacent bicarbon- ate molecule (Fig. 1). The only other water molecules at the active site vicinity are two H2O molecules hydrogen bonded to the oppo- site side of the bicarbonate molecule. The lack of hydrogen bonds at the CO2 binding site of 2-KPCC aids in discrimination between H2O and CO2 which is vital to the function of the enzyme (Scheme 1). In mechanistic terms, this prevents the irreversible production of acetone that could occur upon protonation (Scheme 3), rather than carboxylation of the ketopropyl cleavage product. Other enzymes with more hydrogen bonds to CO2 are not constrained by having to discriminate against solvent. For example, for plants to successfully catalyze CO2 fixation and pre- vent photorespiration, the enzyme 1,5-bisphosphate carboxylase/ oxidase (Rubisco) must discriminate against binding relatively hydrophobic O2 rather that H2O at the CO2 site (for a review, see [24]). Thus, Rubisco utilizes a strongly polarizing, hydrophilic CO2 binding site without compromising catalytic efficiency due to un- wanted side reactions with H2O. 2-KPCC utilizes the inverse strat- egy to promote carboxylation over protonation.

Although the structure of CO2-bound 2-KPCC was solved by uti- lizing the reverse physiological reaction (Scheme 4), the position of

CO2 at the active site may provide insight as to how the forward reaction (Schemes 1 and 2) and catalysis of acetoacetate formation take place. Superposition of the CO2-bound structure on the 2-K-S- CoM substrate bound structure reveals a favorable position of CO2 with respect to the ketopropyl moiety for electrophilic addition and the carbon atom of CO2 resides 4.2 Å from methylene carbon of the ketopropyl moiety of 2-K-S-CoM (Fig. 3A). This correlates 14 14 with CO2 exchange experiments where C is incorporated at the C1 of acetoacetate during carboxylation [4]. Carboxylation at this position could potentially place the carboxylate group of ace- toacetate at the alternate anion binding site, competing with the Fig. 3. (A) Superimposition of the CO2 bound structure (carbons colored green) on CoM-SH sulfonate for Arg-365 and promoting release of products. the 2-K-S-CoM substrate bound structure (carbons colored cyan; PDB ID: 1MO9). H2O molecules are depicted as spheres (red) and the histidine orientated H2O Interestingly, in the 2-K-S-CoM substrate bound structure [7] a his- molecule from the substrate bound structure is colored magenta. Hydrogen bond tidine oriented H2O molecule within hydrogen bonding distance of distances are given in angstroms. The arrow represents potential electrophilic the carbonyl oxygen stabilizes the enolate intermediate from the addition of CO2 to the C1 of the ketopropyl group of 2-K-S-CoM. (B) Unique metal ketopropyl group of 2-K-S-CoM. This interaction is not observed stabilized loop region in the CO2 bound structure with residues Ala-430 and Leu- 431 projecting into the intersubunit space assisting in creating a transitional in the CO2 bound structure due to the absence of this H2O molecule hydrophobic/hydrophilic environment at the active site. CO2, 2-K-S-CoM, bicar- along with a concomitant 180 rotation of the imidazole ring of the ° bonate, and H2O molecules are shown at the active site and alternate anion binding histidine residue. site along with neighboring residues Arg-365 and Arg-56. A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464 463

Porphyridium purpureum [27]. The bicarbonate molecule at the 4.4. The reaction mechanism of 2-KPCC alternate binding site of 2-KPCC has similar bridging H2O mole- cules, although with fewer interactions with protein residues. Crystallographic confirmation of the CO2 binding site presented The only close contact between bicarbonate and 2-KPCC is with herein, combined with ligand-bound and native structures of 2- the amide nitrogen of Gln-509 (3.1 Å) and amine nitrogen of Arg- KPCC [7,14] along with biochemical studies [4] completes a struc- 365 (3.6 Å), which forms a part of the alternate anion binding site ture-based carboxylation mechanism for the enzyme (Fig. 4). To along with the Phe-501 and His-506. initiate the cycle, NADPH binds to oxidized 2-KPCC (Fig. 4, step 1). NADPH subsequently reduces FAD, which in turn reduces the 4.3. The proline flanked loop redox active disulfide (Cys-87–Cys-82) and forms a covalent bond to the of Cys-87 (Fig. 4, steps 2 and 3). The FAD-thiol complex As described in a previous structure [7], a proline flanked loop is collapses to an FAD-thiolate charge transfer complex and the sub- displaced markedly on substrate binding so as to enclose substrate strate 2-K-S-CoM enters into the active site through an open chan- 2-K-S-CoM in the active site (Fig. 1). This loop is part of a larger nel formed by the walls of two subunits of the dimer, which closes loop that extends to Met-438, and a hexacoordinated metal ion upon substrate binding (Fig. 4, step 4) [7]. A hydrophobic channel (Fig. 2B–D) stabilizes it so that residues Pro-420 and Pro-432 are forms upon substrate binding and is stabilized at the base of the re- brought into close proximity. This causes the hydrophobic side gion by two cis-proline residues and a metal binding site. CO2 con- chains of Ala-430 and Leu-431 to project into the subunit interface sequently enters the channel and binds in a hydrophobic pocket where CO2 is bound, making a hydrophobic pocket quite close to strategically aligned to facilitate electrophilic attack at atom C1 of the hydrophilic site where bicarbonate is bound (Fig. 3B). This the ketopropyl group of 2-K-S-CoM (Fig. 4, step 5). This is facilitated lends support to the proposal that CO2 and bicarbonate exist in through nucleophilic attack of the redox-active distal cysteine thiol equilibrium after decarboxylation of acetoacetate. Interestingly, on the C1–S bond, resulting in a mixed disulfide. Base stabilization

Leu-431 is also one of the proposed ligands for the metal ion itself. of the resulting ketopropyl enolate (by a histidine-ligated H2O

Fig. 4. The reaction mechanism of 2-KPCC derived from the previously proposed mechanism [7] and substrate-free (PDB ID 1MOK), substrate-bound (PDBID 1MO9), mixed- disulfide substrate-bound (PDB ID 2C3C), and CO2-bound crystal structures. Structure figures were generated in PyMOL from the crystal structures and accompany the ChemDraw sketches for the individual steps. 464 A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464

molecule) with concomitant attack of the CO2 molecule on atom C1 ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the of 2-K-S-CoM produces acetoacetate, regenerating CoM-SH for an- Xanthobacter strain Py2 epoxide carboxylase system. Biochemistry 39, 1294– 1304. other round of catalysis (Fig. 4, step 6). The acetoacetate carboxyl- [5] Argyrou, A. and Blanchard, J.S. (2004) Flavoprotein disulfide reductases: ate moiety is stabilized at the alternate anion binding site by two advances in chemistry and function. Prog. Nucl. Acid Res. Mol. Biol. 78, 89– 142. H2O molecules, Gln-509, and Arg-365, which also interact with [6] Pai, E.F. (1991) Variations on a theme: The family of FAD-dependent NAD(P)H- the Co-MSH sulfonate moiety. This hydrogen bonding competition (disulphide)-oxidoreductases. Curr. Opin. Struct. Biol. 1, 796–803. with Arg-365 promotes release of products CoM-SH and acetoace- [7] Nocek, B., Jang, S.B., Jeong, M.S., Clark, D.D., Ensign, S.A. and Peters, J.W. (2002) tate with collapse of the disulfide bond to complete the cycle. Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M In summary, the crystal structure of 2-KPCC observed in a state oxidoreductase/carboxylase. Biochemistry 41, 12907–12913. showing the presence of CO2 and 2-K-S-CoM provides the first [8] Begley, T.P. and Ealick, S.E. (2004) Enzymatic reactions involving novel mechanisms of carbanion stabilization. Curr. Opin. Chem. Biol. 8, 508–515. visualization of CO2 bound to 2-KPCC, helping complete a struc- [9] Cleland, W. (1998) The chemistry of carbanions and carbanion-like transition ture-based catalytic mechanism for CO2 fixation by 2-KPCC. The states in: Comprehensive Biological Catalysis (Sinnott, M., Ed.), pp. 1–6, unique binding mode of CO2 at the hydrophobic subunit interface Academic Press. [10] Kappel, W.K. and Olson, R.E. 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