STUDIES ON THE CONFORMATIONAL-CHANGES IN THE BACTERIAL-CELL WALL BIOSYNTHETIC ENZYME UDP-N-ACETYLGLUCOSAMINE ENOLPYRUVYLTRANSFERASE (MURA)

The enzyme UDP-N-acetylglucosamine (UDP-GlcNAc) enolpyruvyltransferase (MurA), the target of the antibiotic fosfomycin, was investigated by small-angle X-ray scattering (SAXS) and fluorescence spectroscopy to d etect conformational changes that had been proposed on the basis of th e crystal structure of unliganded and liganded MurA. The SAXS data ind icate that binding of UDP-GlcNAc to free enzyme results in substantial conformational changes, which can be interpreted as the transition fr om an open to a closed form. Fosfomycin did not affect the structure o f free enzyme or sugar-nucleotide-bound MurA. Phosphoenolpyruvate (pyr uvate-P) appeared to induce a structural change upon addition to free enzyme, which differed from that observed upon binding of UDP-GlcNAc. Fluorescence experiments were performed using the hydrophobic fluoresc ence probe 8-anilino-1-naphthalene sulfonate (ANS). The fluorescence q uenching of MurA/ANS solutions upon addition of UDP-GlcNAc of pyruvate -P was concentration dependent in a saturating manner, yielding appare nt dissociation constants of Kd(UDP-GlcNAc) = 59 mu M and Kd(pyruvate- P) = 240 mu M. The results suggest that binding of substrates does not exclusively follow an ordered mechanism with UDP-GlcNAc binding first , although binding of UDP-GlcNAc to free enzyme is preferred and possi bly influenced by pyruvate-P. The reaction thus appears to follow an i nduced-fit mechanism, in which the binding site for fusfomycin, and pr esumably also for pyruvate-P, is created by the interaction of free en zyme with the sugar nucleotide. The methods described here provide a t ool for the characterization of site-directed mutants of MurA and the interaction of this enzyme with potential inhibitors.