Publications

• 1H, 13C and 15N NMR assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae.

  Mo H., Dai Y., Pochapsky S.S., Pochapsky T.C.

  J Biomol NMR 14:287-288(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• XAS investigation of the structure and function of Ni in acireductone dioxygenase.

  Al-Mjeni F., Ju T., Pochapsky T.C., Maroney M.J.

  Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy- ... >> More

  Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy-3-keto-5-methylthiopentene) with Fe-ARD and dioxygen produces formate and 2-keto-4-methylthiobutanoic acid, the alpha-ketoacid precursor of methionine. Ni-ARD reacts with acireductone and dioxygen to produce methylthiopropionate, CO, and formate and does not lie on the methionine recycle pathway. An X-ray absorption spectroscopy (XAS) study of the structure of the catalytic Ni center in resting Ni-ARD enzyme and the enzyme-substrate complex is reported. This study establishes the structure of the Ni site in resting Ni-ARD as containing a six coordinate Ni site composed of O/N-donor ligands including 3-4 histidine residues, demonstrates that the substrate binds to the Ni center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand, and provides insight into the mechanism of catalysis employed by a Ni-containing dioxygenase. Efficiently relaxed and hyperfine-shifted resonances are observed in the (1)H nuclear magnetic resonance spectrum of Ni-ARD that can be attributed to the His imidazoles ligating the paramagnetic Ni ion and are consistent with the XAS results regarding His ligation. These resonances show significant perturbation in the presence of substrate, confirming that the metal ion interacts directly with the substrate. << Less

  Biochemistry 41:6761-6769(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae.

  Dai Y., Pochapsky T.C., Abeles R.H.

  Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the s ... >> More

  Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate. << Less

  Biochemistry 40:6379-6387(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A bacterial enzyme that catalyzes formation of carbon monoxide.

  Wray J.W., Abeles R.H.

  We have isolated and purified an enzyme (E-2) from Klebsiella pneumoniae, which catalyzes the formation of CO from CH3-S-CH2-CH2-CO-C(OH) = CH-O-(III). This compound is an intermediate in the conversion of 5'-methylthioadenosine to methionine. Concomitant with CO formation, methylthiopropionic aci ... >> More

  We have isolated and purified an enzyme (E-2) from Klebsiella pneumoniae, which catalyzes the formation of CO from CH3-S-CH2-CH2-CO-C(OH) = CH-O-(III). This compound is an intermediate in the conversion of 5'-methylthioadenosine to methionine. Concomitant with CO formation, methylthiopropionic acid and formate are produced and O2 is consumed. E-2 also catalyzes the formation of CO, formate, and butyrate from CH3-CH2-CH2-CO-C(OH) = CH-O-(IIIa), the desthio analog of III. Experiments with isotopic IIIa have shown that formate is derived from 1-C, and CO from 2-C. E-2 has a M(r) = 18,500 and requires Mg2+, and no chromophoric cofactor has been detected. << Less

  J. Biol. Chem. 268:21466-21469(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• One protein, two enzymes.

  Dai Y., Wensink P.C., Abeles R.H.

  Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2' are separable on an anion exchange colu ... >> More

  Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2' are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity. E-2' activity is obtained when Fe2+ is added. Production in intact E. coli of E-2 and E-2' depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity. << Less

  J. Biol. Chem. 274:1193-1195(1999) [PubMed] [EuropePMC]

• Modeling and experiment yields the structure of acireductone dioxygenase from Klebsiella pneumoniae.

  Pochapsky T.C., Pochapsky S.S., Ju T., Mo H., Al-Mjeni F., Maroney M.J.

  Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on th ... >> More

  Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on the type of metal ion bound in the active site. The solution structure of nickel-containing ARD (Ni-ARD) was determined using NMR methods. X-ray absorption spectroscopy, assignment of hyperfine shifted NMR resonances and conserved domain homology were used to model the metal-binding site because of the paramagnetism of the bound Ni2+. Although there is no structure in the Protein Data Bank within 3 A r.m.s deviation of that of Ni-ARD, the enzyme active site is located in a conserved double-stranded b-helix domain. Furthermore, the proposed Ni-ARD active site shows significant post-facto structural homology to the active sites of several metalloenzymes in the cupin superfamily. << Less

  Nat. Struct. Biol. 9:966-972(2002) [PubMed] [EuropePMC]

• Intermediates in the conversion of 5'-S-methylthioadenosine to methionine in Klebsiella pneumoniae.

  Furfine E.S., Abeles R.H.

  Extracts of Klebsiella pneumoniae oxidatively convert 1-phospho-5-S-methylthioribose (1-PMTR) to alpha-keto-gamma-methylthiobutyrate, a precursor of methionine, and to S-methylthiopropionate and formate. One equivalent of formate is produced per equivalent of alpha-keto-gamma-methylthiobutyrate an ... >> More

  Extracts of Klebsiella pneumoniae oxidatively convert 1-phospho-5-S-methylthioribose (1-PMTR) to alpha-keto-gamma-methylthiobutyrate, a precursor of methionine, and to S-methylthiopropionate and formate. One equivalent of formate is produced per equivalent of alpha-keto-gamma-methylthiobutyrate and two equivalents of formate per equivalent of methylthiopropionate. Two compounds were identified as intermediates in this reaction sequence: 1-phospho-5-S-methylthioribulose (1-PMT-ribulose) and 1-phospho-2,3-diketo-5-S-methylpentane. The enzyme, 1-PMTR isomerase, which converts 1-PMTR to 1-PMT-ribulose was highly purified. In addition, a protein fraction was isolated which converts 1-PMT-ribulose to the phosphodiketone. A second protein fraction was isolated that converts the phosphodiketone to an intermediate which has not been isolated so far. This intermediate is oxidatively converted to alpha-keto-gamma-methylthiobutyrate and S-methylthiopropionate by a third protein fraction. Methylthiopropionate is not derived from free alpha-keto-gamma-methylthiobutyrate. << Less

  J Biol Chem 263:9598-9606(1988) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The methionine salvage pathway in Klebsiella pneumoniae and rat liver. Identification and characterization of two novel dioxygenases.

  Wray J.W., Abeles R.H.

  The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella ... >> More

  The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniae and rat liver. This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the alpha-keto acid precursor of methionine). Previously, we had purified an enzyme (E2) from Klebsiella which catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously. This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III. Experiments with 18O2 show that E2 is a dioxygenase which incorporates one molecule of 18O into formate and butyric acid. No cofactor has been identified. We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine. The keto acid is probably produced non-enzymically in Klebsiella. We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III. This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified. Experiments with 18O2 show that E3 is a dioxygenase which incorporates an 18O molecule into formate and the alpha-keto acid. In rat liver CO formation was not detected. << Less

  J Biol Chem 270:3147-3153(1995) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.