Crystallization and preliminary X-ray analysis of PaaAC, the main component of the hydroxylase of the Escherichia coli phenylacetyl-coenzyme A oxygenase complex

The expression, purification, crystallization and preliminary crystallographic analysis of the PaaAC complex is reported. This is the main component of the E. coliphenylacetyl-coenzyme A oxygenase complex. The Escherichia coli paa operon encodes enzymes of the phenylacetic acid-utilization pathway that metabolizes phenylacetate in the form of a coenzyme A (CoA) derivative. The phenylacetyl-coenzyme A oxygenase complex, which has been postulated to contain five components designated PaaABCDE, catalyzes ring hydroxylation of phenylacetyl-CoA. The PaaAC subcomplex shows low sequence similarity to other bacterial multicomponent monooxygenases (BMMs) and forms a separate branch on the phylogenetic tree. PaaAC, which catalyzes the hydroxylation reaction, was purified and crystallized in the absence of a bound ligand as well as in complexes with CoA, 3-hydroxybutyryl-CoA, benzoyl-CoA and the true substrate phenylacetyl-CoA. Crystals of the ligand-free enzyme belonged to space group P2₁2₁2₁ and diffracted to 2.65 Å resolution, whereas complexes with CoA and its derivatives crystallized in space group P4₁2₁2 and diffracted to ∼2.0 Å resolution. PaaAC represents the first crystallized BMM hydroxylase that utilizes a CoA-linked substrate