FEBS Letters 585 (2011) 459–464

journal homepage: www.FEBSLetters .org

Structural basis for carbon dioxide binding by 2-ketopropyl coenzyme Moxidoreductase/carboxylase q

Arti S. Pandey a, David W. Mulder a, Scott A. Ensign b, John W. Peters a,⇑a Department of Chemistry and Biochemistry and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, MT 59717, United Statesb Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, United States

a r t i c l e i n f o

Article history:Received 1 November 2010Revised 17 December 2010Accepted 20 December 2010Available online 27 December 2010

Edited by Stuart Ferguson

Keywords:Disulfide oxidoreductaseCarboxylaseCarbon dioxideAcetoacetateCoenzyme M

0014-5793/$36.00 � 2011 Federation of European Biodoi:10.1016/j.febslet.2010.12.035

Abbreviations: 2-KPCC, 2-ketopropyl coenzyme MFAD, flavin adenine dinucleotide; NADP, nicotinamidphate; 2-K-S-CoM, 2-ketopropyl coenzyme M; CoM-toethanesulfonate); DSOR, disulfide oxidoreductasedeviation; Rubisco, 1,5-bisphosphate carboxylase/oxid

q Coordinates and structure factors have been deposunder the accession code 3Q6J.⇑ Corresponding author. Fax: +1 406 994 7212.

E-mail address: john.peters@chemistry.montana.e

a b s t r a c t

The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been deter-mined in a state in which CO2 is observed providing insights into the mechanism of carboxylation.In the substrate encapsulated state of the enzyme, CO2 is bound at the base of a narrow hydrophobicsubstrate access channel. The base of the channel is demarcated by a transition from a hydrophobicto hydrophilic environment where CO2 is located in position for attack on the carbanion of the keto-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.

Structured summary:2-KPCC binds to 2-KPCC by x-ray crystallography (View interaction)

� 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction NADP+-dependent carboxylase that reductively cleaves and car-

Xanthobacter Py2 is one of several microorganisms that can growon ethylene, propylene or butylene as sole carbon and energy source.During aerobic metabolism of short chain alkenes by Xanthobacterautotrophicus Py2, coenzyme M is conjugated to highly reactive,short chain epoxides for subsequent reductive carboxylation tob-keto acids [1–3]. The last step of the pathway is facilitated by2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC), aflavin adenine dinucleotide (FAD)-containing nicotinamide adeninedinucleotide phosphate (NADP)-dependent oxidoreductase. 2-KPCCcatalyzes reversible carboxylation of ketopropyl-CoM thioether(2-K-S-CoM), yielding acetoacetate and regenerating coenzyme M(2-mercaptoethanesulfonate) (CoM-SH) (Scheme 1) [4].

In contrast to the role of CoM-SH as a C1 carrier in methanogen-esis, CoM-SH acts as a C3 carrier throughout multiple steps in theepoxide carboxylation pathway. 2-KPCC is an FAD-containing

chemical Societies. Published by E

oxidoreductase/carboxylase;e adenine dinucleotide phos-SH, coenzyme M (2-mercap-; RMSD, root mean squarease

ited in the Protein Data Bank

du (J.W. Peters).

boxylates a thioether substrate.2-KPCC is a member of the disulfide oxidoreductases (DSOR), a

family of enzymes which catalyze reductive cleavage of a disulfide-bound substrate followed by subsequent protonation of leavinggroups (for a review, see [5]). At the core of DSOR enzymes thereis a highly redox active cysteine pair, separated by 4 residues, atwhich substrate reduction occurs [6]. The reaction catalyzed by2-KPCC is similar to that of other DSORs, but is unique in twoways: (i) 2-KPCC cleaves a thioether rather than a disulfide bond,and (ii) 2-KPCC catalyzes reversible carboxylation. The catalyticmechanism of 2-KPCC was originally proposed to involve a base-stabilized enolate intermediate of acetone (Scheme 2) [4].

Subsequent structural characterization of 2-K-S-CoM bound en-zyme suggested that the catalytic acid for stabilization of the ace-tone enolate is a histidine bound H2O molecule [7]. In contrast withother DSOR mechanisms, which require protonation of the leavinggroup for productive catalysis, protonation would result in dead-end, irreversible production of acetone, which occurs only whenCO2 is not present (Scheme 3) [4].

In the presence of CO2, protonation to form acetone is negligi-ble, and nearly all 2-K-S-CoM is converted into acetoacetate andCoM [4].

The reverse of the physiological reaction, decarboxylation of ace-toacetate in the presence of CoM-SH and NADP+, also presumablyoccurs through the formation of an enolate intermediate of acetoneand yields 2-K-S-CoM (Scheme 4) [4].

lsevier B.V. All rights reserved.

Fig. 1. (A) Cartoon representation of the structure of 2-KPCC observed in a state withsubunit 2, purple) and a unique loop region is colored orange. The transparent surfaceregion displays the active site area as sticks and depicts CO2 bound along with bicarbonato hydrophobicity/hydrophilicity (brown/cyan). The blue mesh represents the 2Fo � Fc maactive site are shown as dashes with distance in angstroms. The residue label color correCO2, bicarbonate, and 2-K-S-CoM: C, green; O, red; S, orange. All figures were generated

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 1.

460 A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464

Stabilization of a carbanion through charge delocalization withan 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 enzymaticcarboxylation mechanisms include Schiff-base formation betweena lysine and pyridoxal phosphate to provide an electron sink[10,11]. This strategy is not used by 2-KPCC, which has an activesite devoid of lysine residues.

By soaking crystals of FAD-containing 2-KPCC with NADP+ andproducts CoM-SH and acetoacetate, we have enabled the reverseof the physiological reaction resulting in catalytic decarboxylationof acetoacetate, producing the substrates 2-K-S-CoM and CO2. Thestructure of this complex represents the first direct visualization ofthe mode of CO2 recognition used by 2-KPCC. Comparison of theCO2 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 aidsin discrimination against H2O at the active site.

bound CO2. The overall structure is colored according to subunit (subunit 1, blue;rendering (green) depicts the active site formed at the subunit interface. The zoomte, 2-K-S-CoM and selected neighboring residues with side chains colored accordingp for CO2, bicarbonate and H2O molecules contoured at 1.3r. Hydrogen bonds at the

sponds to the residues contributed by the two subunits. Atomic coloring scheme forwith PyMOL [28].

Fig. 2. (A) Surface representation of the hydrophobic channel where CO2 (ball andstick representation) enters the enzyme and resides at its base. (B) Proline flankedloop region (orange) which aids in formation of the CO2 insertion channel. (C and D)Metal binding site at the base of proline flanked loop region. The central atom of theoctahedral binding site is depicted in green and shown coordinated (black dashes)to neighboring residues and H2O molecule (red). The blue mesh (D) represents the2Fo � Fc map contoured at 1.3r and hydrogen bond distance are in angstroms.

A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464 461

2. Materials and methods

2.1. Expression, purification, and crystallization

Expression of X. autotrophicus Py2 cells [12] and purification of2-KPCC [13] were carried out as described previously. Crystalswere obtained by incubating purified 2-KPCC (�30 mg/ml) withacetoacetate, CoM-SH, and NADP+ added in sequential order for10 min prior to addition of an equal amount of precipitating solu-tion of 0.1 M ammonium acetate, 0.085 M sodium citrate pH 5.6,25% PEG 4000 and 30% glycerol. The crystals were grown at roomtemperature using vapor diffusion conditions described previously[14], but reduction with DTT was not performed prior to datacollection.

2.2. Structure determination and refinement

The data were collected from a single cryo-cooled crystal atSSRL beamline 7–1. The raw data were processed with MOSFLM[15] and scaled and merged with the CCP4 program suite [16].Crystals of 2-KPCC belong to the monoclinic space group P21 withone dimer in the asymmetric unit with the following unit cellparameters, a = 88.1 Å, b = 60.0 Å, c = 105.9 Å, and b = 102.4�. Thesubstrate bound crystal structure of 2-KPCC was used as a startingmodel (PDB ID: 1MO9), as the crystals are nearly isomorphous.Iterative cycles of refinement and model building using REFMAC(version 5.5.0109) [17] and COOT [18], respectively, were per-formed until convergence, and the structure was refined to the res-olution of 1.92 Å. Data collection, processing, and refinementstatistics are given in Supplementary Table 1. Superimpositionof the structure on the substrate bound crystal structure of2-KPCC has an overall root mean square deviation (RMSD) valueof 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 moleculein a hydrophobic pocket of one of the subunits adjacent to a newlysynthesized 2-K-S-CoM molecule (Fig. 1), indicating that enzy-matic decarboxylation of acetoacetate has occurred. Previous workhas shown that the substrate 2-K-S-CoM becomes encapsulatedwithin the protein due to ligand-induced conformational changes[14]. CO2 refines well into the linear density with limited residualsand strategically resides at the hydrophobic interface between thetwo subunits of the enzyme (Fig. 1). The CO2 molecule has verylimited water interactions, differing from previously determinedCO2 bound structures where hydrogen bonding for the positioningor 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 alocation termed as the alternate anion binding site which was pro-posed earlier to be the site of acetoacetate binding (Fig. 1) [14]. Thealternate anion binding site presumably serves to stabilize the neg-ative charge buildup during acetoacetate formation [14]. On thebasis of the presence of H2O molecules of which positions are con-served in all forms of 2-KPCC solved structures, present withinhydrogen bonding distances to the apical atoms of the trigonal pla-nar density, a bicarbonate molecule was modeled at this site. Ace-tate from the crystallization buffer was ruled out based on the factthat this trigonal planar density is not seen in other crystals of 2-KPCC formed with the same crystallization mix. Two of the oxygenatoms of the bicarbonate molecule make hydrogen bonds to twoH2O molecules (2.7 and 2.9 Å) and the third oxygen atom is 2.6 Å

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 carbonatom of bicarbonate is 2.7 Å away from the carbonyl oxygen ofthe ketopropyl group and 4.6 Å away from the methyl carbon ofthe ketopropyl group.

The presence of CO2 was observed in one subunit of the struc-ture, while the other subunit shows CoM-SH and ambiguous den-sity that may be consistent with bicarbonate and acetone. Becausedecarboxylation of acetoacetate in the presence of CoM-SH andNADP+ is presumed to occur through the formation of an enolateintermediate of acetone (Scheme 4), this conformation could rep-resent an immediate mechanistic decarboxylation step of acetoac-etate without concomitant formation of 2-K-S-CoM and capture ofCO2. Interestingly, the acetone molecule observed in the secondsubunit is stabilized by an H2O molecule which is in turn stabilizedby the carbonyl oxygen of Ala-430, lending support that it could bethe enolate intermediate of acetone decarboxylation product fromacetoacetate. It is not uncommon for crystal structures to have dif-ferent reaction intermediates in the different subunits. As anexample, the crystal structure of a dioxygenase contains three dif-ferent reaction intermediates in the different subunits of thehomotetrameric enzyme [23].

3.2. Hydrophobic channel for CO2 insertion

The resulting 2-KPCC X-ray crystal structure with 2-K-S-CoMand CO2 has a hydrophobic channel leading from bulk solvent tothe hydrophobic CO2 binding pocket adjacent to the active siteand the methylene group of the thioether linkage (Fig. 2A). Thehydrophobic channel is presumably the pathway for CO2 insertion

Fig. 3. (A) Superimposition of the CO2 bound structure (carbons colored green) onthe 2-K-S-CoM substrate bound structure (carbons colored cyan; PDB ID: 1MO9).H2O molecules are depicted as spheres (red) and the histidine orientated H2Omolecule from the substrate bound structure is colored magenta. Hydrogen bonddistances are given in angstroms. The arrow represents potential electrophilicaddition of CO2 to the C1 of the ketopropyl group of 2-K-S-CoM. (B) Unique metalstabilized loop region in the CO2 bound structure with residues Ala-430 and Leu-431 projecting into the intersubunit space assisting in creating a transitionalhydrophobic/hydrophilic environment at the active site. CO2, 2-K-S-CoM, bicar-bonate, and H2O molecules are shown at the active site and alternate anion bindingsite along with neighboring residues Arg-365 and Arg-56.

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prior to carboxylation. Tight closing of a proline flanked loopregion assists in the formation of the hydrophobic CO2 channel.Featuring two proline residues (Pro-420 and Pro-421) in the cisconformation at one of the termini of the region and a proline res-idue (Pro-432) at the other terminus, the region is tightly loopedand structurally unique among DSOR enzymes (Fig. 2B). The regionis further stabilized near its base of one of the termini by an octa-hedral metal binding site from coordination by the carbonyl atomsof Leu-431 (2.5 Å), Val-429 (2.5 Å), Ala-447 (2.4 Å), Leu-427 (3.2 Å),Ser-450 (2.4 Å) and an H2O molecule (2.3 Å) (Fig. 2C and D).Although the identity of the metal could not be determined unam-biguously by ICP-MS, it is thought to be a metal cation such as Mg2+

given the octahedral coordination geometry of the site.

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 interactionsin the form of hydrogen bonds in 2-KPCC that could contribute topolarizing the substrate CO2. CO2 is strategically located at the baseof 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 carbonatom of CO2. There is a single hydrogen bond between an oxygenatom of CO2 and an H2O molecule bridged to an adjacent bicarbon-ate molecule (Fig. 1). The only other water molecules at the activesite vicinity are two H2O molecules hydrogen bonded to the oppo-site side of the bicarbonate molecule. The lack of hydrogen bondsat the CO2 binding site of 2-KPCC aids in discrimination betweenH2O and CO2 which is vital to the function of the enzyme(Scheme 1). In mechanistic terms, this prevents the irreversibleproduction of acetone that could occur upon protonation(Scheme 3), rather than carboxylation of the ketopropyl cleavageproduct. Other enzymes with more hydrogen bonds to CO2 arenot constrained by having to discriminate against solvent. Forexample, for plants to successfully catalyze CO2 fixation and pre-vent photorespiration, the enzyme 1,5-bisphosphate carboxylase/oxidase (Rubisco) must discriminate against binding relativelyhydrophobic 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 ofCO2 at the active site may provide insight as to how the forwardreaction (Schemes 1 and 2) and catalysis of acetoacetate formationtake 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 additionand the carbon atom of CO2 resides 4.2 Å from methylene carbonof the ketopropyl moiety of 2-K-S-CoM (Fig. 3A). This correlateswith 14CO2 exchange experiments where 14C is incorporated atthe C1 of acetoacetate during carboxylation [4]. Carboxylation atthis position could potentially place the carboxylate group of ace-toacetate at the alternate anion binding site, competing with theCoM-SH sulfonate for Arg-365 and promoting release of products.Interestingly, in the 2-K-S-CoM substrate bound structure [7] a his-tidine oriented H2O molecule within hydrogen bonding distance ofthe carbonyl oxygen stabilizes the enolate intermediate from theketopropyl group of 2-K-S-CoM. This interaction is not observedin the CO2 bound structure due to the absence of this H2O moleculealong with a concomitant 180� rotation of the imidazole ring of thehistidine residue.

4.2. The bicarbonate binding site of 2-KPCC

Both active sites show the presence of trigonal planar densitymodeled as bicarbonate at the alternate anion binding site pro-posed earlier to be the site of acetoacetate binding [14]. Precedentfor bicarbonate recognition by the alternate ion binding site ap-pears in published structures of b-carbonic anhydrases from Hae-mophilus influenzae and Escherichia coli, where bicarbonatemolecules were found at an inhibitory site independent from theenzyme active site [25]. The authors identified two H2O moleculesbridging the bicarbonate molecule and protein functional groups(PDB ID’s 2ESF & 2A8D). These residues are structurally conservedin b-carbonic anhydrase structures from Pisum sativum [26] and

A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464 463

Porphyridium purpureum [27]. The bicarbonate molecule at thealternate binding site of 2-KPCC has similar bridging H2O mole-cules, although with fewer interactions with protein residues.The only close contact between bicarbonate and 2-KPCC is withthe amide nitrogen of Gln-509 (3.1 Å) and amine nitrogen of Arg-365 (3.6 Å), which forms a part of the alternate anion binding sitealong with the Phe-501 and His-506.

4.3. The proline flanked loop

As described in a previous structure [7], a proline flanked loop isdisplaced markedly on substrate binding so as to enclose substrate2-K-S-CoM in the active site (Fig. 1). This loop is part of a largerloop that extends to Met-438, and a hexacoordinated metal ion(Fig. 2B–D) stabilizes it so that residues Pro-420 and Pro-432 arebrought into close proximity. This causes the hydrophobic sidechains of Ala-430 and Leu-431 to project into the subunit interfacewhere CO2 is bound, making a hydrophobic pocket quite close tothe hydrophilic site where bicarbonate is bound (Fig. 3B). Thislends support to the proposal that CO2 and bicarbonate exist inequilibrium after decarboxylation of acetoacetate. Interestingly,Leu-431 is also one of the proposed ligands for the metal ion itself.

Fig. 4. The reaction mechanism of 2-KPCC derived from the previously proposed mechandisulfide substrate-bound (PDB ID 2C3C), and CO2-bound crystal structures. StructureChemDraw sketches for the individual steps.

4.4. The reaction mechanism of 2-KPCC

Crystallographic confirmation of the CO2 binding site presentedherein, combined with ligand-bound and native structures of 2-KPCC [7,14] along with biochemical studies [4] completes a struc-ture-based carboxylation mechanism for the enzyme (Fig. 4). Toinitiate the cycle, NADPH binds to oxidized 2-KPCC (Fig. 4, step1). NADPH subsequently reduces FAD, which in turn reduces theredox active disulfide (Cys-87–Cys-82) and forms a covalent bondto the thiol of Cys-87 (Fig. 4, steps 2 and 3). The FAD-thiol complexcollapses to an FAD-thiolate charge transfer complex and the sub-strate 2-K-S-CoM enters into the active site through an open chan-nel formed by the walls of two subunits of the dimer, which closesupon substrate binding (Fig. 4, step 4) [7]. A hydrophobic channelforms upon substrate binding and is stabilized at the base of the re-gion by two cis-proline residues and a metal binding site. CO2 con-sequently enters the channel and binds in a hydrophobic pocketstrategically aligned to facilitate electrophilic attack at atom C1 ofthe ketopropyl group of 2-K-S-CoM (Fig. 4, step 5). This is facilitatedthrough nucleophilic attack of the redox-active distal cysteine thiolon the C1–S bond, resulting in a mixed disulfide. Base stabilizationof the resulting ketopropyl enolate (by a histidine-ligated H2O

ism [7] and substrate-free (PDB ID 1MOK), substrate-bound (PDBID 1MO9), mixed-figures were generated in PyMOL from the crystal structures and accompany the

464 A.S. Pandey et al. / FEBS Letters 585 (2011) 459–464

molecule) with concomitant attack of the CO2 molecule on atom C1of 2-K-S-CoM produces acetoacetate, regenerating CoM-SH for an-other round of catalysis (Fig. 4, step 6). The acetoacetate carboxyl-ate moiety is stabilized at the alternate anion binding site by twoH2O molecules, Gln-509, and Arg-365, which also interact withthe Co-MSH sulfonate moiety. This hydrogen bonding competitionwith Arg-365 promotes release of products CoM-SH and acetoace-tate with collapse of the disulfide bond to complete the cycle.

In summary, the crystal structure of 2-KPCC observed in a stateshowing the presence of CO2 and 2-K-S-CoM provides the firstvisualization of CO2 bound to 2-KPCC, helping complete a struc-ture-based catalytic mechanism for CO2 fixation by 2-KPCC. Theunique binding mode of CO2 at the hydrophobic subunit interfacepromotes reversible carboxylaton/decarboxylation. The CO2 bind-ing properties of 2-KPCC contrast with Rubisco, the most abundantprotein on earth, which activates CO2 at a highly polar site to pro-mote photosynthesis over photorespiration from the hydrophobicsubstrate O2.

Acknowledgments

This work was supported by National Institutes of Health GrantGM51805 to S.A.E., by Department of Energy Grant DE-FG02-04ER15563 to J.W.P., and the NASA Astrobiology Institute (NAI)-funded NNA08C-N85A Astrobiology Biogeocatalysis ResearchCenter to J.W.P. The authors also acknowledge funding for theestablishment and operation of the Environmental and BiofilmMass Spectrometry Facility at Montana State University (MSU)through the Defense University Research Instrumentation Program(DURIP, Contract Number: W911NF0510255) and the MSU Ther-mal Biology Institute from the NASA Exobiology Program (ProjectNAG5-8807). Portions of this research were carried out at theStanford Synchrotron Radiation Laboratory (SSRL), a national userfacility operated by Stanford University on behalf of the US Depart-ment of Energy, Office of Basic Energy Sciences. The SSRL StructuralMolecular Biology program is supported by the US Department ofEnergy, Office of Biological and Environmental Research, the USNational Institutes of Health, National Center for Research Re-sources, Biomedical Technology program, and the US NationalInstitute of General Medical Sciences.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2010.12.035.

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