This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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