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dc.contributor.authorRehm, BHA
dc.contributor.authorAntonio, RV
dc.contributor.authorSpiekermann, P
dc.contributor.authorAmara, AA
dc.contributor.authorSteinbuchel, A
dc.date.accessioned2018-04-20T03:42:40Z
dc.date.available2018-04-20T03:42:40Z
dc.date.issued2002
dc.identifier.issn0167-4838
dc.identifier.doi10.1016/S0167-4838(01)00299-0
dc.identifier.urihttp://hdl.handle.net/10072/373496
dc.description.abstractA threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281–D290, A372–C382, E578–A589 and V585–A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaCPa) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaCRe) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaCPa-1-265-PhaCRe-289-589) showed 13% of wild type in vitro activity with the fusion point located at a surface-exposed loop region.
dc.description.peerreviewedYes
dc.languageEnglish
dc.publisherElsevier BV
dc.relation.ispartofpagefrom178
dc.relation.ispartofpageto190
dc.relation.ispartofissue1
dc.relation.ispartofjournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
dc.relation.ispartofvolume1594
dc.subject.fieldofresearchBiochemistry and Cell Biology not elsewhere classified
dc.subject.fieldofresearchBiological Sciences
dc.subject.fieldofresearchcode060199
dc.subject.fieldofresearchcode06
dc.titleMolecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: In vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.hasfulltextNo Full Text
gro.griffith.authorRehm, Bernd


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