Dithiol- and NAD-dependent degradation of epoxyalkanes by Xanthobacter Py2

Weijers, C.A.G.M.; Jongejan, H.; Franssen, M.C.R.; Groot, de Ae.; Bont, de J.A.M.

doi:10.1007/bf00171961

Cited by 30 publications

(33 citation statements)

References 26 publications

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“…This proposal is strengthened by the recent identification of component II as an NADPH:disulfide oxidoreductase (12). Interestingly, in the absence of CO 2 , epoxide carboxylase catalyzes the isomerization of aliphatic epoxides to form ketones at rates comparable to those observed for carboxylation (4,9) as shown in Equation 3.…”

mentioning

confidence: 95%

“…With respect to cofactor requirements, in vitro epoxide carboxylation requires a source of reductant (DTT, 1 other dithiols, or NADPH) and an oxidant (NAD ϩ ) (4,9). These cofactor requirements are unprecedented for all other carboxylases that have been characterized.…”

mentioning

confidence: 99%

“…With respect to catalytic mechanism, it has been proposed that redox active thiols are involved in epoxide ring opening and the further transformations that form product (9). This proposal is strengthened by the recent identification of component II as an NADPH:disulfide oxidoreductase (12).…”

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confidence: 99%

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Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex from Xanthobacter Strain Py2

Allen

Ensign

1997

Journal of Biological Chemistry

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Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH-and NAD ؉ -dependent carboxylation reaction that forms ␤-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. Xanthobacter strain Py2 is one of several bacteria capable of growth with short chain aliphatic alkenes as carbon and energy sources (1). The first step in alkene metabolism involves an oxidative insertion of an oxygen atom into the olefin bond in a reaction that is catalyzed by an inducible (2), multiprotein (3) alkene monooxygenase as shown for the substrate propylene and product epoxypropane in Equation 1.Epoxides formed in this manner are further metabolized via a novel ring opening and carboxylation reaction that requires CO 2 as a cosubstrate and forms a ␤-keto acid as product as shown in Equation 2 (4, 5).Aliphatic epoxides such as epoxypropane have toxic, mutagenic, and potential carcinogenic properties (6), and their metabolism in bacteria and mammalian systems has been the focus of considerable research in recent years. Epoxide carboxylation as described for Xanthobacter Py2 represents the most recently discovered biological epoxide transformation reaction, the others involving conjugation to glutathione, hydration to dihydrodiols (7), or isomerization to an aldehyde (8). Initial studies of the epoxide-carboxylating enzyme, designated an epoxide carboxylase, indicate that it has cofactor requirements, molecular properties, and a catalytic mechanism as unique as the epoxide carboxylation reaction itself.With respect to cofactor requirements, in vitro epoxide carboxylation requires a source of reductant (DTT, 1 other dithiols, or NADPH) and an oxidant (NAD ϩ ) (4, 9). These cofactor requirements are unprecedented for all other carboxylases that have been characterized. The requirement of oxidant and reductant is intriguing since there is no net redox chemistry involved in epoxide carboxylation. In the course of epoxide carboxylation, there is an apparent transhydrogenation reaction wherein the reductant becomes oxidized and NAD ϩ becomes reduced, although this has not to date been unequivocally demonstrated.With respect to molecular properties, epoxide carboxylase appears to function as a multiprotein complex (10, 11). Fractionation of Xanthobacter cell extracts by anion-exchange chromatography resolved epoxide carboxylase into three fractions, designated fractions I, II, and III based on their order of elution, that could be recombined with restoration of activity (11). The active component of one of these fractions was purified to homogeneity on the basi...

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Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex from Xanthobacter Strain Py2

Allen

Ensign

1997

Journal of Biological Chemistry

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“…1D and E). As noted above, Weijers and et al (1995) have found that in vitro epoxide degradation (isomerization) can be reconstituted in cellfree extracts in the presence of a dithiol and NAD + . These cofactors are also required for in vitro epoxide carboxylation .…”

Section: Introductionmentioning

confidence: 92%

“…The yield of these ketones is approximately 15% relative to the amount of epoxide degraded and, once formed, the ketones are not readily degraded by the cell suspensions . In a separate study, Weijers et al (1995) have found that epoxide degradation activity can be reconstituted in cell-free extracts of Xanthobacter strain Py2 in a reaction that requires the additional presence of NAD + and a reductant (e.g., dithiothreitol). In their studies, a number of 1,2-and 2,3-epoxyalkanes have been stoichiometrically converted to 180 Fig.…”

Section: Introductionmentioning

confidence: 98%

New roles for CO 2 in the microbial metabolism of aliphatic epoxides and ketones

Ensign

Small

Allen

et al. 1998

Archives of Microbiology

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Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO2 in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce beta-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD+ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD+ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO2 as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO2 to organic substrates.

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Hydrolysis of Epoxides

Orru¹,

Faber²

2002

Enzyme Catalysis in Organic Synthesis

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Chiral epoxides and 1,2-diols, which are central building blocks for the asymmetric synthesis of bioactive compounds, can be obtained via the asymmetric hydrolysis of epoxides using enzymesi.e. epoxide hydrolases (EHs) [EC 3.3.2.Xl. Enzymes from mammalian sourcessuch as rat liver tissuehave been investigated in great detail for several decades during detoxification studies L1] ; however, their application for biotransformations on a preparative scale was hampered because of the limited supply of these enzymes, and, as a consequence, the examples reported rarely surpass the millimolar range[*, ' 1. During the past few years, highly selective epoxide hydrolases were identified from a wide range of microbial sources, which allows for an (almost) unlimited supply of these enzymes for preparative-scale applications. These valuable biocatalysts have recently gained considerable attention, and their scope and limitations have been reviewed["*]. Microbial epoxide hydrolases were found to be more abundant than previously expected, and numerous sources, predominantly among bacteria, fungi and (red) yeasts are known to date.The mechanism of enzymatic hydrolysis of epoxides can be compared to that of base-catalysis, i.e. it resembles an SNZ-type opening of the epoxide by the nucleophile (i. e. water), which leads to the formation of the corresponding trans-configurated 1,2-diol. Any chiral center present in the substrate oxirane can be "recognized", thus effecting kinetic resolution or asymmetrization of racemic or meso-epoxides, respectively. The data available to date indicate that the enantioselectivities of enzymes from certain microbial sources can be correlated to the substitutional pattern of various types of substrates: red yeasts (Rhodotorula or Rhodosporidiurn sp.) give best enantioselectivities with monosubstituted oxiranes; hngal cells (e. g. from Aspergillus and Beauveria sp.) are best suited for styrene oxidetype substrates, whereas bacterial enzymes (in particular from Actinornycetes such as Rhodococcus and Nocardia sp.) are the biocatalysts of choice for more highly substituted 2,2-and 2,3-substituted epoxides. In order to overcome the disadvantage of the classic kinetic resolution pattern, i.e. the formation of two enantiomers in each 50 % yield, various deracemization methods based on chemo-enzymatic or purely enzymatic protocols have been developed. The latter led to the highly desirable formation of a single stereoisomer of the diol in 100% theoretical yield. The synthetic potential of epoxide hydrolases for asymmetric synthesis has been proven by the preparation of a number of bioactive compounds.Chiral epoxides and vicinal diols (employed as their corresponding cyclic sulfate or sulfite esters as reactive intermediates) are extensively employed high-value intermediates in the synthesis of chiral compounds because of their ability to react with a broad variety of nucleophiles (Figs. 11.2-1 and 11.2-2). In recent years, extensive efforts have been devoted to the development of chemo-catalytic methods for ...

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Dithiol- and NAD-dependent degradation of epoxyalkanes by Xanthobacter Py2

Cited by 30 publications

References 26 publications

Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex from Xanthobacter Strain Py2

Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex from Xanthobacter Strain Py2

New roles for CO 2 in the microbial metabolism of aliphatic epoxides and ketones

Hydrolysis of Epoxides

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