The influence of purification and protein heterogeneity on the crystallization of p-hydroxybenzoate hydroxylase.
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 C222(1). 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 C222(1) 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 dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results.
Van der Laan
J M
JM
Laboratory of Chemical Physics, University of Groningen, The Netherlands.
Swarte
M B
MB
Groendijk
H
H
Hol
W G
WG
Drenth
J
J
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D010084:Oxidation-Reduction
D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase