Enzymes involved in epoxide degradation in Xanthobacter Py2

J. Swaving

    Research output: Thesisinternal PhD, WU

    Abstract

    <p>Due to the differences in biological activity of the enantiomers of racemic compounds, the use of enantiomerically pure drugs and agrochemicals is very much encouraged. The availability of optically pure synthons for the production of drugs is, therefor, of the utmost importance for the pharmaceutical industry.</p><p>A very versatile intermediate in organic synthesis is the epoxide group. Epoxides very easily undergo stereospecific ring-opening reactions and are, therefor, very useful to function when available in an enantiomerically pure form as synthons in the production of optically pure drugs.</p><p>In recent years, a great deal of research has been devoted to the development of a biocatalytic method to produces these optically pure epoxides. A very promising method for this is the enantioselective degradation of racemic epoxides (Chapter 2). Although, such a method has a yield of at most 50% it still can be an interesting option, because, racemic epoxides are relatively cheap. A very nice example of enantioselective degradation is found in the bacterium <em>Xanthobacter</em> Py2 which is able to enantioselectively degrade a racemic mixture of 2,3-epoxyalkanes. The 2S-enantiomers are degraded completely, resulting in optically pure 2R-epoxyalkanes.</p><p>At the start of the research done on the degradation of epoxyalkanes by <em>Xanthobacter</em> Py2 described in this thesis, only little information was available on how degradation proceeds. From experiments performed in crude extracts of propene grown <em>Xanthobacter</em> Py2 it was thought that ketones were the product of epoxyalkane degradation (later it was shown epoxyalkanes are carboxylated to<img src="/wda/abstracts/BETA.GIF" ALT="beta" border="0"/>-keto acids). Furthermore, it was concluded that the degradation was dependent on NAD and an unknown low molecular mass compound, which could be replaced by dithiol compounds such as dithiothreitol (DTT).</p><p>To study the degradation of epoxyalkanes by <em>Xanthobacter</em> Py2 in more detail it was decided to do complementation studies using <em>Xanthobacter</em> Py2 mutants devoid of epoxyalkane-degrading activity (Chapter 3). Several cosmids were found complementing restoring the ability of the mutants to grow on epoxides. One of these cosmids, pEP9 was conjugated into <em>Xanthobacter autotrophicus</em> GJ10. This strain is not able to grow on 1,2-epoxypropane nor able to degrade the compound. In crude extracts of <em>Xanthobacter autotrophicus</em> GJ10 complemented with the pEP9, however, epoxyalkane-degrading activity was demonstrated, but only after addition of the LMF or DTT, indicating the right genes were cloned.</p><p>Subcloning revealed a 4.8 kb fragment able to complement the mutation. This fragment was sequenced and found to contain four open reading frames which code for proteins of 41690, 7388, 57315 and 26111 Da, respectively. A homology search using the Swiss-Prot protein bank did reveal little or no homology for the first two ORFs. For ORF4, homologies were found with short-chain alcohol dehydrogenases like 3-oxoacyl reductase and glucose 1-dehydrogenase. Interestingly, ORF3 showed significant homology with pyridine nucleotide-disulfide oxidoreductases like mercury (II) reductase, glutathione reductase and with dihydrolipoamide dehydrogenase.</p><p>In Chapter 4 and Chapter 5, the protein encoded by this third open reading frame was investigated in more detail. All consensus primary structures of pyridine nucleotide-disulfide oxidoreductase are present on the ORF3 amino acid sequence but the C-terminal active site. Furthermore, from the amino acid sequence it was deduced that the nucleotide binding site of NAD(P) showed more resemblance with NADP-dependent proteins then with NAD-dependent proteins, indicating that the ORF3 is NADP-dependent. Using this information the involvement of NADP was tested in the conversion of epoxyalkane in dialyzed crude extracts of propene grown <em>Xanthobacter</em> Py2. It was shown that NADPH and NAD <sup>+</SUP>could restore the epoxyalkane degradation, indicating that NADPH is in fact the low molecular mass fraction. The dithiols replacing the low molecular mass fraction are probably able to directly reduce the redox active disulfide bridge on the ORF3 protein. NADPH also reduces this disulfide bridge by passing on the electrons via a FAD, which is bound to the protein.</p><p>The ORF3 protein was purified and shown to be involved in epoxyalkane degradation by fractionating crude extracts of propene grown <em>Xanthobacter</em> Py2 and complementing fractions without the ORF3 protein with the purified protein, thus restoring the epoxyalkane-degrading activity.</p><p>The purified ORF3 protein in this stage called component II, was characterized (Chapter 5). The protein was shown to be a homodimeric protein, each subunit containing a tightly bound FAD. The spectral properties of the FAD were investigated and kinetic studies were performed to characterize the protein.</p><p>Characterization on component II showed this protein to have common themes as well as distinct difference with other pyridine nucleotide-disulfide oxidoreductases. This comparison gave no clear understanding on the substrate of the component II and its action in the degradation of epoxides.</p><p>Because the component II protein is missing the C-terminal His-Glu dyad active site found in lipoamide dehydrogenases and glutathione reductases, the protein has a very low activity towards dithiols, therefore the substrate of component II may not even be a dithiol compound. 1,2-Epoxypropane is not a substrate for component II, nor is there any interaction with the compound. The involvement of an other (hypothetical) protein, ORF2 ENCODED, is suggested.</p><p>Finally, in Chapter 6, a possible mechanism for 1,2-epoxypropane degradation is discussed.</p>
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    Supervisors/Advisors
    • de Bont, J.A.M., Promotor
    • de Kok, A., Promotor, External person
    Award date10 Jun 1998
    Place of PublicationS.l.
    Publisher
    Print ISBNs9789054858560
    Publication statusPublished - 1998

    Keywords

    • microbial degradation

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