No abstract

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 59-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 818 relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.