Abstract
UDP-N-acetylmuramyl pentapeptide is an essential component of the cell wall in bacteria. The synthesis of this essential glycopeptide involves a total of eleven gene products; all of which are targets for the development of new and novel antibiotics. We have undertaken the study of the substrate and product interactions with the second enzyme in this pathway, UDP-Nacetylglucosamine- enoylpyruvate reductase (MurB) from E. coll. Using optical spectroscopy, we demonstrate that the bound oxidized fiavin of MurB was rapidly reduced in the presence of 200 #M NADPH. However, in the presence of dioxygen, the enzyme was reoxidized, presumably via the formation of a fiavin-peroxy intermediate. The reoxidized enzyme showed the characteristic FAD optical spectrum, but when compared to the native spectrum, the reoxidized fiavin absorbance is red shifted by 8 nm. The red shifted spectrum is a result of the interaction of NADP+ with the enzyme. Optical difference spectroscopy showed a change in the absorbance at 456,478 and 512 nin. A Kd (NADP+) = 5.8 ± 0.4 /-M was measured. Nicotinamide mononucleotide and AI)P-ribose did not cause this spectral shift, indicating the requirement for the intact NADP+ molecule. The same spectral shift was observed with the substrate UDP-N-acetylglucosamine-enoylpyruvate, indicating that both NADP+ and UDP-N-acetylglucosamine-enoylpyruvate perturb the active-site flavin in the same way. These results are consistent with a common active site pocket for both NADPH and UDP-NAc-glucosamine-enolpyruvate.
Original language | English |
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Pages (from-to) | A1308 |
Journal | FASEB Journal |
Volume | 11 |
Issue number | 9 |
State | Published - 1997 |