Structural Basis for CO<sub>2</sub> Fixation by a Novel Member of the Disulfide Oxidoreductase Family of Enzymes, 2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase<sup>,</sup>

Nocek, B.; Jang, Se Bok; Jeong, Mi Suk; Clark, Daniel D.; Ensign, Scott A.; Peters, John W.

doi:10.1021/bi026580p

Cited by 24 publications

(45 citation statements)

References 38 publications

(50 reference statements)

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“…The inhibition constants reflecting binding to the ES complex (K ii ) were slightly higher, although similar in magnitude to, the K is values. These results support the results of x-ray crystallographic studies of R-HPCDH and 2-KPCC showing that the hydroxypropyl and ketopropyl moieties, in addition to the sulfonate of CoM, are important in high affinity substrate binding (5,28). For methyl-CoM reductase, where the group bound to CoM is the nonpolar methyl group, the ethanesulfonate could be expected to be the major binding determinant, perhaps helping to explain why BES is a much higher affinity inhibitor for this enzyme.…”

Section: Discussionsupporting

confidence: 87%

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Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Boyd

Clark

Kofoed

et al. 2010

Journal of Biological Chemistry

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The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys 82 ) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys 87 was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UVvisible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys 82 . These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.The bacterial metabolism of epoxides formed from shortchain aliphatic alkene epoxidation occurs by a three-step metabolic pathway that uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) 3 as a nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation (Fig.

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Section: Discussionsupporting

confidence: 87%

“…1) (1-3). Epoxide catabolism from (R)-and (S)-epoxypropane converges at the intermediate 2-ketopropyl-CoM (2-KPC), which is a substrate for 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), a unique member of the disulfide oxidoreductase (DSOR) family of enzymes (3)(4)(5).…”

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Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Boyd

Clark

Kofoed

et al. 2010

Journal of Biological Chemistry

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“…It has been proposed that this hydrogen-bonding network is responsible for stabilizing enolacetone formed from thioether bond cleavage, as the interchange thiol attacks the thioether bond, as shown in Fig. 3B (22,28). An additional novel feature of the active site of 2-KPCC is a pair of methionine residues (M140 and M361), which flank the substrate 2-KPC.…”

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confidence: 99%

“…2C, 2-KPCC catalyzes the reduction of a thioether rather than a disulfide bond, a feature not seen in any other known DSOR enzyme. Mechanistic (10) and structural (28,30,31) studies have provided evidence for a reaction mechanism where thioether bond cleavage results in the formation of a mixed disulfide between CoM and the interchange thiol with the formation of the enolacetone anion (Fig. 2C).…”

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confidence: 99%

“…Significant insights into these features have come from X-ray crystallographic structures of 2-KPCC determined for various states, including the substrate-free enzyme (28), a 2-KPCbound form of the enzyme (28), a form of the enzyme where the mixed disulfide of CoM was trapped (31), and a form in which the substrate CO 2 is bound (30). Collectively, the structural characterization of 2-KPCC revealed an activesite architecture unique among DSOR enzymes, where substrate binding induces a conformational change that creates a hydrophobic pocket that encapsulates the substrate 2-KPC (22,28). Of relevance to the question of how 2-KPCC stabilizes the enolacetone anion for subsequent carboxylation is that a hydrogen-bonding network was identified in the 2-KPC-bound enzyme, consisting of an ordered water molecule hydrogen bonded to both the carbonyl oxygen of 2-KPC and two histidine residues (H137 and H84) (Fig.…”

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Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

et al. 2011

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NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK a value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK a value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.The bacterial metabolism of gaseous propylene by the proteobacterium Xanthobacter autotrophicus Py2 and the actinomycete Rhodococcus rhodochrous B276 is initiated by the insertion of a single oxygen atom into the olefin bond of propylene, forming (R)-and (S)-epoxypropane (19,27,35,39). These epoxypropane enantiomers are further metabolized by a three-step linear pathway that uses four enzymes and the atypical cofactor coenzyme M (CoM) (2-mercaptoethanesulfonic acid) to catalyze the net carboxylation of epoxypropane to form acetoacetate as shown in Fig. 1 (1, 2 , 4, 5, 18, 24). NADPH:2-ketopropyl-coenzyme M oxidoreductase/ carboxylase (2-KPCC) is the CO 2 -fixing enzyme of this pathway, catalyzing the reductive cleavage and carboxylation of 2-ketopropyl-CoM (2-KPC) with the stoichiometry ...

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Substitution of a conserved catalytic dyad into 2‐KPCC causes loss of carboxylation activity

et al. 2016

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The characteristic His-Glu catalytic dyad of the disulfide oxidoreductase (DSOR) family of enzymes is replaced in 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) by the residues Phe-His. 2-KPCC is the only known carboxylating member of the DSOR family and has replaced this dyad potentially to eliminate proton-donating groups at a key position in the active site. Substitution of the Phe-His by the canonical residues results in production of higher relative concentrations of acetone versus the natural product acetoacetate. The results indicate that these differences in 2-KPCC are key in discriminating between carbon dioxide and protons as attacking electrophiles.

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Structural Basis for CO₂ Fixation by a Novel Member of the Disulfide Oxidoreductase Family of Enzymes, 2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase^,

Cited by 24 publications

References 38 publications

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

Substitution of a conserved catalytic dyad into 2‐KPCC causes loss of carboxylation activity

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Structural Basis for CO2 Fixation by a Novel Member of the Disulfide Oxidoreductase Family of Enzymes, 2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase,

Cited by 24 publications

References 38 publications

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

Substitution of a conserved catalytic dyad into 2‐KPCC causes loss of carboxylation activity

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Structural Basis for CO₂ Fixation by a Novel Member of the Disulfide Oxidoreductase Family of Enzymes, 2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase^,