ABSTRACT
The enzyme p-hydroxybenzoate hydroxylase (PHBH) catalyzes the hydroxylation of its substrate in an NADPH-dependent reaction. Crystals of PHBH were routinely grown as the PHBH-substrate complex. Crystals of other catalytic intermediates were obtained by soaking procedures. Crystals of the enzyme-product complex were obtained by conversion of bound substrate into product by enzymatic catalysis inside the crystals, combined with soaking. Attempts to remove the substrate from the crystals of the PHBH-substrate complex by soaking have been unsuccessful because the crystals become too fragile to handle or simply crack. The PHBH-substrate complex could be crystallized in the presence of 60 mM Adenosine 5'-diphosphoribose and 1.0 M sodium dithionite. The overall conformation of the PHBH molecule was found to be very similar to the conformation of PHBH in the crystal structure of the other complexes. Crystallization of PHBH in the presence of monovalent anions, such as chloride or iodide, gave some useful crystals.
