Crystallographic studies of FAD bound form of salicylate hydroxylase from Pseudomonas putida S-1

Research Reactor Institute, Kyoto University* Department of Microbiology, School of Pharmacy, Hokuriku University** Department of Chemistry, Faculty of Science, Kanazawa University*** Department of Chemistry, Graduate School of Science, Kyoto University****
â—‹Yoshihiko Watanabe* Akiko Kita* Kuniharu Ohnishi** Kenzi Suzuki*** Kunio Miki**** Yukio Morimoto*

Salicylate (o-hydroxybenzoate) hydroxylase from Pseudomonas putida S-1, consisting of 421 amino acid residues, catalyzes the decarboxylative hydroxylation of salicylate to catechol and CO2, with a 1:1:1 stoichiometry. It is a flavin-dependent monooxygenase containing 1 mol of FAD per mol of enzyme with a molecular weight of 54,000. The enzyme is a unique monooxygenase that catalyzes the hydroxylation with decarboxylation of salicylate or deformylation of salicylaldehyde. To understand the reaction mechanism and the substrate recognition system, it is important to determine the crystal structure of salicylate hydroxylase.
We have reported the crystallization of apo-form salicylate hydroxylase previously(1). We report here the crystallographic studies of FAD bound form (holo-form) of salicylate hydroxylase from Pseudomonas putida S-1. The crystals of holo-form enzyme were obtained by vapor diffusion method using ammonium sulfate as a precipitant and 2-methyl-2,4-pentanediol as an additive reagent at the room temperature. The crystal was grown to a maximal size of 0.7 x 0.3 x 0.3 mm. The crystals belong to hexagonal space group of P62 or P64, with unit-cell dimension of a=b=141.7 Å, c=62.0 Å. The crystals diffracted X-ray beyond 2.7 Å resolution. Assuming one molecule per asymmetric unit, the crystal volume per unit molecular mass, Vm, is calculated to be 3.3 Å3/Da. The crystal structure analysis is in progress. The comparison of the structures between apo- and holo- enzyme will be discussed.
(1) T. Yabuuchi, K. Suzuki, T. Sato, K. Ohnishi, E. Itagaki, and Y. Morimoto, J. Biochem, 119, 829 (1996).