Complementation of Xanthobacter Py2 mutants defective in epoxyalkane degradation, and expression and nucleotide sequence of the complementing DNA fragment

Abstract

Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkanedegrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified 'low molecular mass fraction (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M I 0 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar t o the wild-type activity. Surprisingly, M10 transformed with complementing cosmid PEP9 showed a constitutively expressed epoxyal kane-degrading activity, which was not observed in the wild-type strain. The cosmid PEP9 was conjugated into Xanthobacter autotrophicus GJIO, which is not able to degrade 1,2-epoxypropane. In crude extracts of X. autotrophicus GJ1 O(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb EcoRI-Hindlll fragment t o be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.Keywords : Xanthobacter Py2, epoxyalkane degradation, complementation INTRODUCTIONSeveral methods have been described to produce optically pure epoxides by biological methods (Weijers et al., 198813; Leak e t a/., 1992;De Bont, 1993). One such method is the enantioselective degradation of racemic epoxides; however, such a process yields a maximum of 50% product. Xantbobacter Py2 was isolated on propene as sole carbon and energy source (Van Ginkel & De Bont, 1986). This strain is able to degrade the (2s)-forms of trans-2,3-epoxybutane and 2,3-epoxypentane, resulting in optically pure (2R)-2,3-epoxyalkanes ; the C,-C, 1,2-epoxyalkanes are degraded completely (Weijers et al., 1988a The EMBL accession number for the sequence reported in this paper is X79863.Ketones have been identified as degradation products of epoxyalkane metabolism in crude extracts of propenegrown cells (Fig. 1). It has been demonstrated that both N A D and an unidentified ' low molecular mass fraction ' (LMF) are involved in the reaction. In a reaction mechanism proposed by Weijers et al. (1994) it was suggested that the LMF is involved in the reduction of the epoxyalkanes to secondary alcohols since the LMF can be replaced by reducing compounds like DTT and other dithiol compounds. The presence of N A D is necessary for the oxidation of the alcohols to the corresponding ketones.Attempts to purify the epoxyalkane-degrading enzyme have not been successful. Consequently, we have now investigated the genetics of epoxyalkane degradation in Xantbobacter Py2. Eventually, we hope to be able to obtain the enzyme to study further its reaction mechanism.In this study we report the selection and characterization of mutants devoid of epoxyalkane-degrading activity, the complementation o...

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

“…within the 4.8-kb fragment of DNA that complemented Xanthobacter mutants incapable of degrading epoxides (16). These results would suggest that a wild type copy of orf5 was still present in the mutant strains that could be complemented.…”

“…As mentioned earlier, a fragment of Xanthobacter DNA with four ORFs complements mutant Xanthobacter strains unable to metabolize epoxides (16). It is clear from the results of three recent studies that component II is the product of the orf3 gene (10 -12).…”

“…These results would suggest that a wild type copy of orf5 was still present in the mutant strains that could be complemented. Notably, not all of the epoxide degradation minus mutants could be complemented with the 4.8-kb fragment of DNA; possibly, these mutants had mutations in orf5 as well (16).…”

“…epoxyalkane-degrading activity. 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).…”

“…The Nterminal sequence of component IV was determined and, as shown in Table V, does not match that of orf4 (Table V). After accessing the sequence of the complementary DNA fragment as deposited by Swaving and coworkers (16) in the European Molecular Biology Library (EMBL accession number X79863), we found that an additional 2.2 kb of DNA beyond the initial 4.8 kb reported in the paper had been sequenced. The additional 2.2 kb of DNA contains three additional ORFs (orf5-7).…”