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

Kofoed, Melissa A.; Wampler, David; Pandey, Arti; Peters, John W.; Ensign, Scott A.

doi:10.1128/jb.05231-11

Cited by 12 publications

(14 citation statements)

References 38 publications

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“…The standard reaction mixture contained 100 m m Tris pH 7.4, 0.2 m m NADPH, 5 m m 2‐KPC, and ±60 m m KHCO 3 with varied [2‐KPCC] (from 0.1 to 0.25 mg). The differences in activities measured by the GC and UV/vis coupled assay from NADPH oxidation assay (Table ) can be attributed to the use of DTT as opposed to the physiological reductant, NADPH .…”

Section: Methodsmentioning

confidence: 99%

“…Second, the resulting base‐stabilized enolacetone intermediate has two possible enzymatic fates: carboxylation to form the desired product acetoacetate, or protonation to form the metabolically unproductive product acetone (Fig. ) . In the presence of saturating CO 2 , carboxylation of the enolacetone intermediate and acetoacetate formation is the predominant catalytic outcome .…”

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confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

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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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“…A pK a Ͼ9.0 was determined, reflecting the hydrophobic environment of this active site ( Fig. 3) (17). NADP ϩ is critical both for stabilizing the C4a adduct and for catalysis (16).…”

Section: The Catalytically Active Reduced Form Of 2-kpcc Is Neither mentioning

confidence: 99%

“…In all cases the total volume of enzyme, DTT, and BES added was less than 4% the total reaction volume and hence did not heavily CO 2 -fixing flavoenzyme influence the solution 1 H/ 2 H composition. The samples were allowed to incubate at 20°C for 4 h. To remove the BES, samples were treated with DOWEX resin as previously (17). Each sample was assayed for activity via addition of the BES-treated enzyme to a 5 mM 2-KPC and 0.1 mM NADPH solution made in 100 mM GTP buffer at pH 7.…”

Section: Proton Inventory Through Solvent Isotope Effectsmentioning

confidence: 99%

The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

Streit

Mattice

Prussia

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma Banerjee NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO 2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO 2. The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pK a between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO 2 fixation.

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“…35 References for the occurrence of other thiols; glutathione amide, 47 mycothiol, 48, 49 bacillithiol, 50, 51 trypanothione and glutathionylspermidine, 52 glutathionylspermidine in E. coli, 53 ergothioneine, 54–56 CoM-SS-CoB, 57, 58 and CoM. 59, 60 …”

Section: Figurementioning

confidence: 99%

The Orphan Protein Bis-γ-glutamylcystine Reductase Joins the Pyridine Nucleotide Disulfide Reductase Family

Kim

Copley

2013

Biochemistry

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Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still “orphan” enyzmes whose activity is known but the genes that encode them have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with mis-assigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine (γ-Glu-Cys) is the major low molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by NanoLC electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in E. coli and re-folded from inclusion bodies. The enzyme had robust GCR activity, but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologs of GCR, suggesting that there is more to be learned about the low molecular weight thiols used in halobacteria.

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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

Cited by 12 publications

References 38 publications

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

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

The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

The Orphan Protein Bis-γ-glutamylcystine Reductase Joins the Pyridine Nucleotide Disulfide Reductase Family

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