Taniguchi Kazuo, Nakamura Akio, Tsurubuchi Kazue, O'Hara Koji, Sawai Tetsuo
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
FEMS Microbiol Lett. 2004 Mar 19;232(2):123-6. doi: 10.1016/S0378-1097(03)00961-3.
Macrolide 2'-phosphotransferase (MPH(2')) catalyzes the transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolide antibiotics. In this study, H198 and H205, conserved in the ATP-binding region motif 1 in the putative amino acid sequence of MPH(2')II, were replaced by Ala to investigate their role. H205 was also subsequently replaced by Asn. H198A and H205N mutant enzymes retained more than 50% of the specific activity of the original enzyme to substrate oleandomycin. On the other hand, the specific activity of the H205A mutant enzyme was reduced to less than 1% of that of the wild enzyme. The results suggested that H205 is crucial for maintaining the catalytic activity of MPH(2')II, and Asn can substitute for His at this position.
大环内酯2'-磷酸转移酶（MPH(2')）催化ATP的γ-磷酸基团转移至大环内酯类抗生素的2'-羟基上。在本研究中，将MPH(2')II假定氨基酸序列中ATP结合区域基序1里保守的H198和H205替换为丙氨酸，以研究它们的作用。随后H205也被替换为天冬酰胺。H198A和H205N突变酶对底物竹桃霉素的比活性保留了原始酶的50%以上。另一方面，H205A突变酶的比活性降低至野生型酶的1%以下。结果表明，H205对于维持MPH(2')II的催化活性至关重要，且天冬酰胺可在该位置替代组氨酸。
