A novel type of pyridine nucleotide-disulfide oxidoreductase is essential for NAD+- and NADPH-dependent degradation of epoxyalkanes by Xanthobacter strain Py2

Abstract

Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD ؉ and dithiols. This multicomponent system has now been fractionated. A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed. This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation. NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion.Propene-grown Xanthobacter strain Py2 (15) contains an enzyme system capable of degrading epoxyalkanes, which are metabolites arising from alkenes by the action of alkene monooxygenase. Recently, both the monooxygenase (23) and the epoxide-degrading system of the organism have received considerable attention. Initially, this interest was based on applied aspects because the organism may be used in the degradation of chlorinated alkenes and epoxides (3, 4, 7, 10) and in the production of optically pure epoxides (18). As it turns out, the epoxide-degrading enzyme system has very intriguing properties and was therefore investigated in detail.Initially, epoxide degradation was studied at the whole-cell level (9, 18). Recently, Weijers et al. (19) were able to demonstrate enzyme activity in extracts if both NAD ϩ and a lowmolecular-mass fraction were included in the assay system. Furthermore, they showed that the low-molecular-mass fraction can be replaced by a range of artificial dithiol compounds, such as dithiothreitol (DTT). Ketones were the product formed under their assay conditions. Allen and Ensign (1) also studied the fate of epoxides in extracts. They included carbonate in their assay system and proposed that the enzyme system of Xanthobacter strain Py2 carboxylated 1,2-epoxyalkanes to form ␤-keto acids. In their view, ketones are a dead-end product which is formed only when carbonate is limiting. The formation of either product from an epoxide is redox neutral. The requirement for both NAD ϩ and a dithiol therefore suggests that reduction of the epoxide is followed by oxidation or vice versa.The first report of a successful fractionation of the epoxidedegrading enzyme system was by Leak and coworkers (Imperial College, London, United Kingdom). They were able to devise a method resulting in two fractions, both of which were required to reconstitute an active epoxide-degrading system (16).Swaving et al. (13) reported the cloning of a 4.8-kb DNA fragment required for complementation of mutants of Xanthobacter strain Py2 defective in epoxide degradation. From the deduced amino acid sequences of the four open reading frames of this fragment no clear information on how the degradation of epoxides proceeds could be gained. However, the protein encoded by ORF3 was of great interest because of its homology to the family of pyridine nucleotide-disulfide oxidoreductases (22). The homology was of special interest because most members of this family use dithiols as a substrate, whereas dithiol...

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

“…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.…”

“…The sequence of this DNA fragment revealed four ORFs, designated orf1, orf2, orf3, and orf4, that may represent one or more of the components of the epoxide carboxylase system (16). Analysis of the molecular weight, N-terminal sequence, and amino acid composition of purified component II revealed that this protein corresponded to orf3 of the complementary DNA fragment (11,12). To determine whether the three epoxide carboxylase components purified in this study correlate with any of the remaining ORFs, the molecular weights, N-terminal sequences, and amino acid compositions of purified components I, III, and IV were determined (Tables V and VI).…”

“…These same cofactors are required for reconstituting epoxide carboxylase activity using the purified protein components. NADPH has been proposed to serve as the physiological reductant for the system based on the characterization of component II as a specific NADPH:disulfide oxidoreductase (12). It is important to note that there is no net redox chemistry involved in the carboxylation of aliphatic epoxides to ␤-keto acids.…”

“…This protein, designated component II since it was purified from fraction II from the initial separation, was characterized as a dimeric flavoprotein consisting of identical 57-kDa sub-units (11). In a separate study, this flavoprotein was shown to possess NADPH:disulfide oxidoreductase activity, suggesting that it is involved in the oxidation of the reductant necessary for epoxide carboxylation (12). Based on the specificity of the flavoprotein, NADPH rather than a cellular dithiol was proposed to serve as the physiological reductant for epoxide carboxylation (12).…”

“…In a separate study, this flavoprotein was shown to possess NADPH:disulfide oxidoreductase activity, suggesting that it is involved in the oxidation of the reductant necessary for epoxide carboxylation (12). Based on the specificity of the flavoprotein, NADPH rather than a cellular dithiol was proposed to serve as the physiological reductant for epoxide carboxylation (12).…”