Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.Oxalate oxidase (OXO; EC 1.2.3.4) 6 catalyzes the oxidation of oxalate, reducing dioxygen to hydrogen peroxide and forming 2 mol of carbonOxalate oxidase is widespread in nature and has been found in bacteria (4), fungi (1, 5), and various plant tissues (6). It has been detected in barley seedling roots during germination and in the leaves of mature barley plants in response to powdery mildew infection (6, 7), suggesting a role in plant signaling and defense. The enzyme has been purified to homogeneity from barley seedling roots and its N-terminal sequence determined, allowing the corresponding cDNA to be isolated and the complete primary sequence to be determined (3,8). These developments led to the recognition that the enzyme, OXO, is identical to an important marker of grain development during germination of wheat called germin (3,8). OXO is a member of a functionally diverse protein superfamily known as the cupins (9) or double stranded ␤-helix proteins (10). Barley OXO forms a hexamer that has extreme stability to heat and proteolysis (11).Spectroscopic studies demonstrated that OXO requires manganese for catalysis (12) and subsequent crystallographic studies on the barley enzyme revealed the structure of the hexamer (Fig. 1a) and confirmed the presence of a mononuclear manganese center buried deep within its jellyroll ␤-barrel domain (13). The manganese is bound by the side chains of three histidines and one glutamate residue, as well as two water molecules that occupy adjacent positions in the roughly octahedral metal complex (Fig. 1b). Based on the lack of obvious optical absorption and the presence of a characteristic EPR spectrum, the manganese ion has been assigned as the reduced Mn(II) oxidation state in the resting enzyme (12). Spectroscopic studies using recombinant OXO expressed in Pichia pastoris confirmed the presence of Mn(II) in the resting recombinant enzyme and provided the first spectroscopic evidence for oxalate binding to the manganese (14). The EPR signal of the anaerobic substrate complex, like that of the nativ...

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.

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