Hoppensack A, Rehm B H, Steinbüchel A
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, D-48149 Münster, Germany.
J Bacteriol. 1999 Mar;181(5):1429-35. doi: 10.1128/JB.181.5.1429-1435.1999.
The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated. Four beta-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 panD gene from Escherichia coli, encoding L-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 from Escherichia 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-encoded L-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 to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of pan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]beta-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.
对来自真养产碱菌（Ralstonia eutropha）的聚羟基丁酸酯（PHA）合酶经4-磷酸泛酰巯基乙胺进行的假定翻译后修饰进行了研究。分离出了4株真养产碱菌HF39的β-丙氨酸营养缺陷型Tn5诱导突变体，其中两个插入位点定位在一个与大肠杆菌panD基因有高度相似性的开放阅读框中，该基因编码L-天冬氨酸-1-脱羧酶（EC 4.1.1.15）；而另外两个插入位点定位在一个与NAD(P)+转氢酶（EC 1.6.1.1）α1亚基有高度相似性的开放阅读框（ORF）中，该亚基由大肠杆菌的pntAA基因编码。通过对真养产碱菌Q20的panD突变体进行互补克隆得到了panD基因。对panD基因区域（3312 bp）进行DNA测序，发现一个365 bp的开放阅读框，其编码的蛋白质与大肠杆菌和枯草芽孢杆菌的PanD氨基酸序列相似性分别为63%和67%。仅将该开放阅读框亚克隆到载体pBBR1MCS-3和pBluescript KS-中，分别导致了真养产碱菌和大肠杆菌SJ16的panD突变体得到互补。通过酶活性测定进一步证实了panD编码的L-天冬氨酸-1-脱羧酶。在panD上游，发现了一个与大肠杆菌pntAA有高度相似性的开放阅读框，其编码NAD(P)+转氢酶亚基α1；在panD下游，鉴定出了两个与pntAB和pntB有高度相似性的开放阅读框，分别编码NAD(P)+转氢酶的α2和β亚基。因此，在真养产碱菌中发现了一种迄今未确定的pan和pnt基因组织形式。使用真养产碱菌的一个panD突变体和[2-14C]β-丙氨酸进行的标记实验没有提供证据表明真养产碱菌PHA合酶通过4-磷酸泛酰巯基乙胺的翻译后共价连接进行修饰，大肠杆菌的panD突变体也未显示出来自真养产碱菌的功能性PHA合酶有可检测到的标记。
