The influence of purification and protein heterogeneity on the crystallization of p‐hydroxybenzoate hydroxylase

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Abstract
The structure of the enzyme p‐hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53‐73] with crystals belonging to space group C2221. Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron‐blue‐purified enzyme appears to be heterogeneous with respect to aggregation state and Cys‐116 oxidation. Most importantly, it could be firmly established that C2221 crystals can only be grown from purely dimeric p‐hydroxybenzoate hydroxylase possessing an intact SH group. Ion‐exchange chromatography on DEAE‐Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dis‐sociating the enzyme oligomers into dimers; sulfite especially gives excellent results.