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dc.contributor.authorHoppensack, A
dc.contributor.authorRehm, BHA
dc.contributor.authorSteinbuchel, A
dc.date.accessioned2018-04-20T04:12:42Z
dc.date.available2018-04-20T04:12:42Z
dc.date.issued1999
dc.identifier.issn0021-9193
dc.identifier.urihttp://hdl.handle.net/10072/373512
dc.description.abstractThe postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutrophaby 4-phosphopantetheine was investigated. Four β-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panDgene from Escherichia coli, encodingL-aspartate-1-decarboxylase (EC 4.1.1.15), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene fromEscherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS− led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encodedL-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity topntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization ofpan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]β-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha.
dc.description.peerreviewedYes
dc.languageEnglish
dc.publisherAmerican Society for Microbiology
dc.publisher.urihttp://jb.asm.org/content/181/5/1429.short
dc.relation.ispartofpagefrom1429
dc.relation.ispartofpageto1435
dc.relation.ispartofissue5
dc.relation.ispartofjournalJournal of Bacteriology
dc.relation.ispartofvolume181
dc.subject.fieldofresearchBiochemistry and Cell Biology not elsewhere classified
dc.subject.fieldofresearchBiological Sciences
dc.subject.fieldofresearchAgricultural and Veterinary Sciences
dc.subject.fieldofresearchMedical and Health Sciences
dc.subject.fieldofresearchcode060199
dc.subject.fieldofresearchcode06
dc.subject.fieldofresearchcode07
dc.subject.fieldofresearchcode11
dc.titleAnalysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from Ralstonia eutropha: Generation of β-Alanine Auxotrophic Tn5 Mutants and Cloning of the panD Gene Region
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
dc.description.versionPublished
gro.rights.copyright© 1999 American Society for Microbiology. The attached file is reproduced here in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.
gro.hasfulltextFull Text
gro.griffith.authorRehm, Bernd


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