A Study of p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

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Abstract
The purification procedure for p-hydroxybenzoate hydroxylase was modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex-Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40-50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (M) of 43,000-45,000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature-dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but 5 SH groups. In the native state of the enzyme only, 1 SH group is accessible to N-ethylmaleimide or 5,5''-dithiobis(2-nitrobenzoic acid). The isoelectric point of the enzyme was 5.8.