Hendricks J K, Mobley H L
Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore 21201, USA.
J Bacteriol. 1997 Sep;179(18):5892-902. doi: 10.1128/jb.179.18.5892-5902.1997.
Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.
幽门螺杆菌脲酶在酶活性位点需要镍离子来发挥催化活性。因此，镍离子必须由该细菌主动获取。幽门螺杆菌的NixA（高亲和力镍转运蛋白）缺陷型突变体仍保留显著的脲酶活性，这表明存在其他镍转运体。对幽门螺杆菌基因组核苷酸序列的分析揭示了NikD的一个同源物，NikD是大肠杆菌中一种依赖ATP的镍转运系统的组成部分。基于该序列，从幽门螺杆菌基因组DNA中通过PCR扩增出一个378 bp的DNA片段，并用作探针来鉴定携带这些序列的幽门螺杆菌λZAPII基因组文库克隆。通过测序揭示了4个分别为621、273、984和642 bp的开放阅读框（abcABCD），预测的多肽分子量分别为22.7、9.9、36.6和22.8 kDa。36.6 kDa的多肽（AbcC）与大肠杆菌ABC转运系统的一种ATP结合蛋白成分具有显著同源性（氨基酸序列同一性为56%），而其他推定蛋白与现有数据库中的多肽均无显著同源性。为了确定这些基因对脲酶活性的可能贡献，分别用卡那霉素抗性（aphA）盒对abcC和abcD进行插入失活，并在幽门螺杆菌UMAB41中构建每个基因的等位基因交换突变体。abcD突变导致脲酶活性降低88%，降至27±31 μmol NH₃/分钟/毫克蛋白（P<0.0001），而nixA和abcC的双突变体导致脲酶活性几乎完全丧失（双突变体中为1.1±1.4 μmol NH₃/分钟/毫克蛋白，而亲本中为228±92 μmol NH₃/分钟/毫克蛋白[P<0.0001]）。然而，脲酶脱辅基酶的合成不受任何abc基因突变的影响。我们得出结论，除了nixA外，abc基因簇也参与了具有催化活性的脲酶的产生。
