X-ray crystal structures of the S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine at 1.8-A resolution.
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 A 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 FADH2 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.
Benson
T E
TE
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
C T
CT
Hogle
J M
JM
D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry
D014537:Uridine Diphosphate N-Acetylglucosamine; C038277:UDP-N-acetylglucosamine-enolpyruvate; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase