Crystal Structure Of The Nosiheptide Resistant Methlytransferase Of Streptomyces Actuosus

Antibiotics play significant roles in the battle against pathogenic microorganism in human history and ever saved countless lives.However, as the broader application, abusing of antibiotics is becoming increasingly serious problem which followed by dangerous drug resistance. Thus the research in the molecular mechanism of microorganism drug resistance is extremely important. Nosiheptide is a kind of widely used antibiotic which is produced by a microogranism named Streptomyces actuosus,which is insensitive to the substance. Streptomyces actuosus bears a gene which codes for the resistant enzyme of rRNA methyltransferase NSR. The protein NSR is able to neutralize nosiheptide by methylating the A1067 at the antibiotic binding site of 23S rRNA on the 50S subunit of ribosome. Nevertheless, the crystal structure of NSR and the detailed enzymatic mechanism remain unknown.In this work, NSR was expressed, purified and crystallized.Finally we obtained both crystals of single NSR and complex of NSR and SAM which were suitable for data collection.The single protein of NSR and NSR in complex with SAM structures were determined at 1.9 and 2.25,respectively.Based on the crystal structures, comprehensive mutants were applied to systematically investigate the functions of NSR.The crystal structure and biochemical analysis show that NSR forms a functional homodimer, indicating that dimerization is required for methyltransferase.NSR is comprised of N terminal RNA binding domain (NTD) and C terminal catalytic domain (CTD).NTD possess a RNA binding domain while CTD has the ability of transfer the methyl group of SAM to the RNA substrate. Thus both the functional areas needs to be investigated.We designed a serious of mutants to test their in vitro activities based on the analysis of each functional area of the structure.The result revealed the two loops are essential for enzymatic activity.