2.2Å STRUCTURE: OF D84E MUTANT OF PORPHOBILINOGEN DEAMINASE. N. C. Picken¹, R. Lambert², S. Awan³, P. M. Jordan², S. P. Wood², ¹Department of Crystallography, Birkbeck College, Malet St., London, ²Department of Biochemistry, University of Southampton, Southampton ³Institute of Ophthalmology, University College London, UK

Porphobilinogen deaminase (PBGD) is the third enzyme in the biosynthetic pathway of tetrapyrroles. PBGD catalyses the stepwise polymerization of four molecules of the substrate porphobilinogen forming the highly unstable intermediate preuroporphyrinogen.

The structure of native E.coli PBGD has been solved to 1.7Å. However a flexible loop region of this structure remains invisible. Therefore it was decided to try freezing crystals in order to try to gain more information about this missing loop.

Asp 84 is a catalytically important residue in the active site cleft. This residue hydrogen bonds to the pyrrole nitrogens of the cofactor and facilitates deamination and stabilizes the developing positive charge throughout the reaction. The D84E mutant of this protein retains 1% of its catalytic activity whereas other mutants, such as D84A and D84N, are catalytically inactive. This makes this mutant an interesting mutant for structural studies.

Crystals of the D84E mutant have been grown and successfully frozen for data collection. A 2.2Å data set was collected and processed. The protein was found to have crystallized in space group P2₁2₁2, which is the same space group as the original structure, however the unit cell of a=84.97 b=75.09 c=48.29 [[alpha]]=[[beta]]=[[gamma]]=90deg. is significantly smaller than that of the native crystal. This shrinkage in the unit cell was assumed to be a result of freezing the crystal.

Due to the shrinkage in the unit cell molecular replacement methods had to be used before any structural refinement could take place.