Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: Generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region

The postulated posttranslational modification of the polyhydroxybutyrate (P HA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigat ed. Four beta-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 w ere isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding L-aspart ate-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)(+) tra nshydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli. The panD gene was cloned by complementation of the panD m utant 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 s equence similarity to PanD from E. coli and Bacillus subtilis, respectively . Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS- l ed to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encoded L-aspartate-1-decarboxylase was further confirme d by an enzymatic assay. Upstream of panD, an ORF,vith strong similarity to pntAA from E, coli, encoding NAD(P)(+) transhydrogenase subunit alpha 1 wa s found; downstream of panD, two ORFs with strong similarity to pntAB and p ntB, encoding subunits alpha 2 and beta of the NAD(P)(+) transhydrogenase, respectively, were identified, Thus, a hitherto undetermined organization o f pan and pnt genes was found in R. eutropha. Labeling experiments using on e of the R. eutropha panD mutants and [2-C-14]beta-alanine provided no evid ence that R. eutropha PHA synthase is covalently modified by posttranslatio nal attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exh ibit detectable labeling of functional PHA synthase from R. eutropha.