Leimkühler S, Kern M, Solomon P S, McEwan A G, Schwarz G, Mendel R R, Klipp W
Ruhr-Universität Bochum, Fakultät für Biologie, Lehrstuhl für Biologie der Mikroorganismen, Germany.
Mol Microbiol. 1998 Feb;27(4):853-69. doi: 10.1046/j.1365-2958.1998.00733.x.
Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.
通过随机Tn5诱变，分离出14株不能以黄嘌呤作为唯一氮源生长的荚膜红细菌突变体。其中5个Tn5插入位点位于两个相邻的染色体EcoRI片段内，这些片段与根据真核黄嘌呤脱氢酶保守氨基酸序列合成的寡核苷酸杂交。对该区域的DNA序列分析揭示了两个开放阅读框，分别命名为xdhA和xdhB，它们编码黄嘌呤脱氢酶。推导的XDHA氨基酸序列包含两个[2Fe-2S]簇和FAD的结合位点，而XDHB预计包含钼蝶呤辅因子。与荚膜红细菌不同，这三个辅因子结合位点存在于真核黄嘌呤脱氢酶的单一多肽链中。荚膜红细菌黄嘌呤脱氢酶的氨基酸序列与真核黄嘌呤脱氢酶的相似程度高于与巨大脱硫弧菌的黄嘌呤脱氢酶相关醛氧化还原酶的相似程度。当添加次黄嘌呤或黄嘌呤作为唯一氮源时，xdhA-lacZ融合蛋白的表达被诱导。nifR1（ntrC）和nifR4（rpoN，编码sigma54）中的突变对xdh基因表达没有影响。一种推测的感知底物可用性的激活剂似乎对黄嘌呤有反应，但对次黄嘌呤没有反应。通过引物延伸分析确定了xdhA的转录起始位点。与已知启动子元件的比较未发现明显同源性。荚膜红细菌的黄嘌呤脱氢酶被纯化至同质。该酶由两个亚基组成，分子量分别为85 kDa和50 kDa。两个亚基的N端氨基酸测序证实了预测的起始密码子。天然酶的分子量测定为275 kDa，表明其为α2β2亚基结构。对荚膜红细菌黄嘌呤脱氢酶的钼辅因子分析表明，它含有钼蝶呤辅因子而非钼蝶呤二核苷酸衍生物。
