GENPAT SOLUTION OF THE STRUCTURE OF PROTOCATECHUATE-3,4-DIOXYGENASE FROM PSEUDOMONAS CEPACIA. Christer E. Nordman, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

The newly developed molecular-replacement procedure GENPAT (Nordman, Acta Cryst. 1994, A50, 68-72) has been used to solve the structure of tetrameric (m.wt. 198,000) protocatechuate-3,4-dioxygenase (PCD) from P. cepacia (Ludwig, Weber & Ballou, J. Biol. Chem. 1984, 259, 14840-14842) using as a model one subunit (m.wt. 48,900) of dodecameric PCD from P. aeruginosa (Ohlendorf, Lipscomb & Weber, Nature, 1988, 366, 403-405). The subunit of PCD from P. aeruginosa contains 438 residues, and that of P. cepacia 431. Both contain one nonheme iron atom. The identity between the two proteins is 48 percent, and there are many deletions and insertions. A conservative search model was used, consisting of 382 residues (1868 atoms) of a polyalanine (glycine) chain, from P. aeruginosa, with many gaps, and representing 1.6 percent of the unit cell contents of the P. cepacia PCD.

P. cepacia PCD crystallizes in P2₁2₁2₁ with four tetramers per cell. Ludwig et al. observed absences of odd orders of , in the hO, diffraction pattern, indicating the presence of a non-crystallographic local 2-fold axis at x ~ 0.25, and approximately parallel to c and suggesting that the local tetramer symmetry is 222. With a tetramer as the asymmetric unit, the rotational search with one monomer would normally give four symmetrically independent solutions. If a noncrystallographic 2-fold axis of the tetramer is parallel to a 2-fold axis in the Patterson, the rotation search solutions reduce to two correspondingly enhanced maxima. Thus the rotation search is facilitated by the orientation of the tetramer 2-fold axis. There is no corresponding enhancement of the maxima in the translation search.

With data to 2.9 Å resolution, the two maxima in the rotation search, mapped as the correlation coefficient, were 0.0809 and 0.0744, or 9.9 and 9.1 [[sigma]], respectively, in terms of standard deviations above the background. Spurious peaks were found at up to 3[[sigma]]; among these were peaks resulting from an approximate 2-fold symmetry within part of the subunit (Ohlendorf et al.). The four symmetrically independent solutions to the translation search were found at 3.2 to 4.1[[sigma]]; the known location of the non-crystallographic 2-fold axis made a fully exhaustive translation search unnecessary.