MurB catalyzes the second committed step in the synthesis of peptidoglycan, a key component of the bacterial cell wall, The crystal structures of both a S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine were solved and refined at 1.8 Angstrom resolution, The single point mutation of residue 229 from serine to alanine eliminated a hydroxyl group which has previously been proposed to play a critical role as a proton donor during the second half-reaction of MurB, namely, reoxidation of FADH(2) and reduction of the enolpyruvyl substrate, The mutation also resulted in the loss of the water molecule-hydrogen bonded to the serine hydroxyl in the wild-type structure changing the hydrogen-bonding network with in the active site, Comparison of the wild-type and S229A mutant structures confirms that the dramatic kinetic defect of an approximately 10(7)-fold decrease observed for the Ser 229 Ala mutant in the second half-reaction [Benson, T, E., Walsh, C. T., & Massey, V. (1997) Biochemistry 36, 796-805] is a direct result of the loss of the serine hydroxyl moiety rather than other nonspecific active-site changes or general structural defects.
