STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF AMINOGLYCOSIDE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的总体目标是获得一对氨基糖苷类耐药酶，链霉素6-磷酸转移酶APH(6)-Ia和-ID的详细结构和功能信息。据预测，这两种酶都会在链霉素链的第6位磷酸化链霉素，从而使其失活。在灰色链霉菌中发现了APH(6)-Ia基因，而在一些植物和人类病原菌中发现了APH(6)-Ia基因。预计这两种酶之间以及与特征良好的APH(3‘)-IIIa酶之间的详细比较将有助于设计对一系列氨基糖苷磷酸转移酶有效的酶抑制剂。该项目的具体目标是：(1)表达和表征APH(6)-Ia和-ID；(2)鉴定参与催化、底物结合和结构域结合的氨基酸残基；以及(3)确定APH(6)-Ia或-ID的X射线结构。今年，在实现项目目标方面取得了一些进展。APH(6)-Ia.获得了一株含有灰色链霉菌生物合成基因簇的革兰氏阳性菌变青链霉菌，该基因含有aph(6)-Ia基因。从培养物中分离出表达载体，并用聚合酶链式反应扩增出aph(6)-Ia基因。以这种方式获得的基因被发现与Genbank公布的aph(6)-Ia序列相差一个核苷酸；通过直接从稻瘟病菌基因组DNA扩增基因并对目的区域进行测序证实了这种差异。这一结果表明，翻译序列中的Ser-262实际上是丙氨酸。将该基因亚克隆到表达载体pET-15b中，并在Rosetta(DE3)pLysS大肠杆菌中表达。SDS-PAGE分析表明，该蛋白主要以不溶性包涵体形式存在，但Western blotting显示也有部分可溶性蛋白存在。现已用镍亲和层析纯化了该可溶性酶。在这一点上，目标是确认纯化的APH(6)-Ia是活性的，并根据生物物理和稳态动力学性质对其进行表征。APH(6)-ID。获得了一个含有aph(6)-ID基因的质粒，该基因最初是从植物致病细菌丁香假单胞菌中分离出来的，但也在人类致病菌株中发现。从该载体上扩增出aph(6)-ID基因，并将其亚克隆到表达载体pET-15b中。与APH(6)-Ia一样，SDS-PAGE和Western blotting分析表明，重组蛋白大部分以包涵体形式存在，但也有部分可溶性蛋白存在。重组蛋白已经纯化，并采取了一些步骤试图获得有活性的酶，尽管这被证明是难以捉摸的。与APH(6)-Ia一样，来年的目标将是获得活性酶并对其进行表征。

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. The overall goal of this project is to obtain detailed structural and functional information about a pair of aminoglycoside resistance enzymes, the streptomycin 6-phosphotransferases APH(6)-Ia and -Id. Both enzymes are predicted to phosphorylate streptomycin at position 6 of its streptidine ring, thereby inactivating it. The gene for APH(6)-Ia is found within producer strain Streptomyces griseus, whereas the gene for APH(6)-Ia has been found in a number of plant and human pathogenic bacteria. It is expected that a detailed comparison between the two enzymes, as well as with the well-characterized enzyme APH(3')-IIIa, will aid in the design of enzyme inhibitors effective against a range of aminoglycoside phosphotransferases. The specific aims of the project are to: (1) Express and characterize APH(6)-Ia and -Id; (2) Identify amino acid residues involved in catalysis, substrate binding, and domain association; and (3) Determine the X-ray structure of either APH(6)-Ia or -Id. This funding year, some progress has been made toward achieving the aims of the project. APH(6)-Ia. A culture of the gram-positive bacterium Streptomyces lividans containing a plasmid with the streptomycin biosynthetic gene cluster from S. griseus, which includes the aph(6)-Ia gene, was obtained. The plasmid was isolated from the culture, and the aph(6)-Ia gene PCR-amplified. The gene obtained in this manner was found to differ from the published Genbank sequence for aph(6)-Ia by one nucleotide; this difference was confirmed by amplifying the gene directly from S. griseus genomic DNA and sequencing through the region of interest. This result indicates that Ser-262 in the translated sequence is actually an alanine. The gene then was subcloned into the expression vector pET-15b, from which the N-terminal His-tagged recombinant fusion protein was expressed in Rosetta(DE3)pLysS E. coli cells. Analysis by SDS-PAGE revealed that most of the protein was present in insoluble inclusion bodies, but Western blotting revealed that some soluble protein was obtained as well. The soluble enzyme now has been purified using nickel affinity chromatography. At this point, the goal is to confirm that the purified APH(6)-Ia is active and to characterize it in terms of biophysical and steady state kinetic properties. APH(6)-Id. A plasmid containing the aph(6)-Id gene originally isolated from the plant pathogenic bacterium Pseudomonas syringae, but also found in human pathogenic strains, was obtained. This aph(6)-Id gene was PCR-amplified from the plasmid and subsequently subcloned into the expression vector pET-15b. As with APH(6)-Ia, SDS-PAGE and Western blotting analysis of expression revealed that most of the recombinant protein was present in inclusion bodies, although some soluble protein was also present. The recombinant protein has been purified and a number of steps have been taken to try to obtain active enzyme, although this has proved elusive. As with APH(6)-Ia, the goal for the upcoming year will be to obtain active enzyme and characterize it.