OXIDATION OF HYDROCYLAMINE BY BACTERIA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 羟胺（NH 2 OH）是细菌如自养氨氧化细菌（AAOB）和甲烷氧化细菌（MOB）氧化氨过程中的中间产物。羟胺是一种有效的诱变剂，并通过各种酶系统快速转化为亚硝酸盐（HNO 2），包括两种不相关的酶，羟胺氧化还原酶（HAO）和细胞色素P460，每种酶在其活性位点具有独特的血红素。 这两种酶都存在于AAOB模型中，欧洲亚硝化单胞菌，尽管HAO比细胞色素P460丰富得多，并且以高得多的速率支持羟胺氧化。虽然这些酶几乎没有直接的医学重要性（尽管其中一种存在于一些机会致病菌中），但它们独特的活性位点结构使它们对所有生物化学家都具有相当大的兴趣，用于研究这些酶的技术可以应用于医学兴趣的酶。其次，HAO是氨氧化成亚硝酸盐的主要原因，亚硝酸盐是全球氮循环的主要成分。虽然HAO在体内氧化羟胺中的作用似乎已经很好地确立，但细胞色素P460的作用不太确定，并且可能参与一氧化氮（NO）的螯合或去除，而不是羟胺氧化。 尽管对N.然而，对于HAO输出和加工的途径以及活性位点血红素交联的机制仍然未知，这在很大程度上是因为HAO酶对于N. europaea的HAO基因，也是由于N. europaea尚未在任何异源宿主细菌中表达。在未发表的实验中，我将N. europaea在质粒上的诱导型lac启动子后面，并将该质粒引入两种细菌物种中。铜绿假单胞菌和大肠杆菌可以产生HAO多肽作为一个包含体，但出口的多肽到周质和插入血红素，包括血红素P460，没有发生。这表明，一个特定的酶或转运蛋白，而不是通常用于c-细胞色素，可能参与HAO成熟。有趣的是，在所有具有HAO基因的细菌中，包括N.例如，在一种植物中，该基因总是跟随着一个功能未知的推定膜蛋白的基因，该基因很可能是HAO转运蛋白或成熟酶（Bergmann et al 2006）。我们目前正在研究这种可能性，将该基因与HAO基因一起放入低拷贝数表达质粒（Novagen的pACYCDuet）中，并尝试在铜绿假单胞菌和大肠杆菌中表达。杆菌这将确定这种未知的膜蛋白是否是必需的和足够的，沿着一般的分泌途径和c-血红素加工基因（如大肠杆菌的ccm基因簇）。coli），用于HAO表达。如果HAO可以在异源宿主细菌中表达，则可以通过定点诱变来修饰HAO基因。特别是，突变体HAO酶没有活性位点酪氨酸可以构建和表征生化（紫外-可见光谱，EPR光谱，羟胺氧化，NO结合或还原和氧化还原滴定血红素）。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hydroxylamine (NH2OH) is an intermediate during the oxidation of ammonia by bacteria such as autotrophic ammonia oxidizing bacteria (AAOB) and methane oxidizing bacteria (MOB). Hydroxylamine is a potent mutagen, and is rapidly converted to nitrite (HNO2) by various enzyme systems, including two unrelated enzymes, hydroxylamine oxidoreductase (HAO) and cytochrome P460, each with a unique a heme at their active sites. Both enzymes are present in the model AAOB, Nitrosomonas europaea, although HAO is much more abundant than cytochrome P460, and supports hydroxylamine oxidation at a much higher rate. Although these enzymes are little direct medical importance (although one of them is present in some opportunistic pathogens), their unique active site structure makes them of considerable interest to all biochemists, and the techniques used to study these enzymes can be applied to enzymes of medical interest. Secondly, HAO is responsible for much of ammonia oxidation to nitrite, a major component of the global nitrogen cycle. While the role of HAO in oxidizing hydroxylamine in vivo appears to be well established, the role of cytochrome P460 is less certain, and may be involved in sequestration or removal of nitric oxide (NO) rather than hydroxylamine oxidation. Despite extensive work on the biochemistry of the HAO of N. europaea, the pathways for HAO export and processing and the mechanism of active site heme cross-linking remain unknown, in large part because the enzyme is essential for the survival of N. europaea and also because the gene for HAO of N. europaea has not been expressed in any heterologous host bacterium. In unpublished experiments, I have placed the HAO gene of N. europaea behind the inducible lac promoter on a plasmid, and introduced the plasmid into two bacterial species. Both Pseudomonas aeruginosa and Escherichia coli could produce the HAO polypeptide as an inclusion body, but the export of the polypeptide into the periplasm and insertion of hemes, including heme P460, did not occur. This suggests that a specific enzyme or transporter, other than that generally used for c-cytochromes, may be involved in HAO maturation. It is interesting to note that, in all bacteria with an HAO gene, including N. europaea, the gene is always followed by a gene for a putative membrane protein of unknown function, which might well be an HAO transporter or maturation enzyme (Bergmann et al 2006). We are currently investigating this possibility by placing the gene into a low copy-number expression plasmid (Novagen's pACYCDuet) together with the gene for HAO, and attempting expression in P. aeruginosa, and E. coli. This will determine if this unknown membrane protein is required and sufficient, along with the general secretory pathway and c-heme processing genes (such as the ccm gene cluster of E. coli) found in most bacteria, for HAO expression. If HAO could be expressed in a heterologous host bacterium, the HAO gene could then be modified by site-directed mutagenesis. In particular, mutant HAO enzymes without the active-site tyrosine could be constructed and characterized biochemically (UV-visible spectroscopy, EPR spectroscopy, hydroxylamine oxidation, NO binding or reduction and redox titrations of hemes).