STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF AMINOGLYCOSIDE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。本项目的总体目标是获得一对氨基糖苷抗性酶——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，这两种酶都能使链霉素链嘌呤环的第6位磷酸化，从而使其失活。APH(6)-Ia基因存在于生产菌株灰色链霉菌（Streptomyces griseus）中，而APH(6)-Ia基因存在于许多植物和人类致病菌中。我们期望通过对这两种酶的详细比较，以及与已被充分表征的APH（3’）-IIIa酶的比较，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。该项目的具体目的是：(1)表达和表征APH(6)-Ia和-Id；(2)识别参与催化、底物结合和结构域结合的氨基酸残基；(3)测定APH(6)-Ia或-Id的x射线结构。本资助年度，在实现该项目的目标方面取得了一些进展。APH ia(6)。我们获得了革兰氏阳性细菌lividans链霉菌的培养物，其质粒含有来自金黄色葡萄球菌的链霉素生物合成基因簇，其中包括aph(6)-Ia基因。从培养物中分离质粒，扩增aph(6)-Ia基因。用这种方法获得的基因与已发表的aph(6)-Ia的Genbank序列有一个核苷酸的差异；这种差异通过直接从金黄色葡萄球菌基因组DNA中扩增基因并通过感兴趣的区域测序得到证实。结果表明，翻译序列中的Ser-262实际上是一个丙氨酸。然后将该基因亚克隆到表达载体pET-15b中，在Rosetta(DE3)pLysS大肠杆菌细胞中表达n端his标记的重组融合蛋白。SDS-PAGE分析显示，大部分蛋白存在于不溶性包涵体中，但Western blotting也发现了一些可溶性蛋白。目前已用镍亲和层析法纯化了可溶性酶。在这一点上，我们的目标是确认纯化的APH(6)-Ia是有活性的，并在生物物理和稳态动力学性质方面对其进行表征。APH (6) id。获得了一种含有aph(6)-Id基因的质粒，该基因原分离自植物病原菌丁香假单胞菌，但也在人类致病菌株中发现。该aph(6)-Id基因从质粒中pcr扩增，随后亚克隆到表达载体pET-15b中。与APH(6)-Ia一样，SDS-PAGE和Western blotting表达分析显示，大部分重组蛋白存在于包涵体中，尽管也存在一些可溶性蛋白。重组蛋白已被纯化，并采取了许多步骤试图获得活性酶，尽管这已被证明是难以捉摸的。与APH(6)-Ia一样，未来一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.