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Using synchrotron radiation, the X-ray diffraction intensities of crystals of
p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate,
were measured to a resolution of 1.9 A. Restrained least-squares refinement
alternated with rebuilding in electron density maps yielded an atom model of the
enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148
reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located.
In the final model, only three residues have deviating phi-psi angle
combinations. One of them, the active site residue Arg44, has a well-defined
electron density and may be strained to adopt this conformation for efficient
catalysis. The mode of binding of FAD is distinctly different for the different
components of the coenzyme. The adenine ring is engaged in three water-mediated
hydrogen bonds with the protein, while making only one direct hydrogen bond with
the enzyme. The pyrophosphate moiety makes five water-mediated versus three
direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen
bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring
also makes only direct hydrogen bonds, but virtually only with main-chain atoms.
The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar
to that in glutathione reductase, while the riboflavin-binding parts of these
two enzymes have no structural similarity at all. The refined 1.9 A structure of
the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further
refinement of the 2.3 A structure of the enzyme-product complex. The result was
a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an
improved geometry. Comparison between the complexes indicated only small
differences in the active site region, where the product molecule is rotated by
14 degrees compared with the substrate in the enzyme-substrate complex. During
the refinements of the enzyme-substrate and enzyme-product complexes, the flavin
ring was allowed to bend or twist by imposing planarity restraints on the
benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed
angle between the benzene ring and the pyrimidine ring was 10 degrees for the
enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because
of the high temperature factors of the flavin ring in the enzyme-product
complex, the latter value should be treated with caution. Six out of eight
peptide residues near the flavin ring are oriented with their nitrogen atom
pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)
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