Crystallization and preliminary crystallographic analysis ofE. coliuridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase in two new crystal forms

Abstract

Uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5'-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 ,~ resolution and shows the symmetry and systematic absences of space group P2j2~2j. These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0,A resolution data set at a synchrotron source showed cell dimensions at ca 100K of a = 51.0, b---79.3 and c ---87.1 ,~, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 A resolution consistent with space group P2~, unit-cell dimensions (ca 100K) a-----50.7, b=92.4, c--85.5A, and /5= 104 ° , and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.