Given the prominence of arginine catabolism in the regulation of diverse metabolic pathways such as ureagenesis 1 and nitric oxide biosynthesis, 2 the synthesis and evaluation of nonreactive arginine analogues as possible enzyme inhibitors or receptor antagonists is a rapidly-growing focus of medicinal chemistry. 2,3 To date, only one enzyme of mammalian arginine catabolism, rat liver arginase, has yielded an X-ray crystal structure to guide structure-based inhibitor design efforts. 4 This trimeric metalloenzyme contains a binuclear manganese cluster in the active site of each subunit required for maximal catalytic activity. 5 Arginine hydrolysis is achieved by a metal-activated solvent molecule that symmetrically bridges the Mn 2+ -Mn 2+ ion pair in the native enzyme. The reaction coordinate of hydrolysis is postulated to proceed through a tetrahedral intermediate resulting from nucleophilic attack of metal-bridging hydroxide ion at the guanidinium carbon of arginine (Figure 1a). 4 The tetrahedral borate anion is a modest, noncompetitive inhibitor of arginase, with K is ) 1.0 mM and K ii ) 0.26 mM; inhibition is even more pronounced in the presence of product ornithine, which is a competitive inhibitor with K i ) 1.0 mM. 6,7 In order to understand the mode of inhibition, we now report the X-ray crystal structure of the ternary arginase-ornithineborate complex. The tetrahedral borate anion mimics binding interactions postulated for the tetrahedral transition state(s) in the catalytic reaction (Figure 1a). 4 This result provides an important foundation for the design, synthesis, and evaluation of the first boronic acid analogue of arginine, 2(S)-amino-6boronohexanoic acid (6). The high affinity of this inhibitor for arginase is proposed to result from the structural similarity between its hydrated form and the proposed tetrahedral intermediate (and flanking transition states) for arginase-catalyzed arginine hydrolysis (Figure 1b).Crystal Structure of the Arginase-Ornithine-Borate Complex. Crystals of rat liver arginase were prepared as described 8 and gradually transferred to a buffer solution containing 10 mM ornithine and 10 mM sodium borate. X-ray diffraction data to 3.0 Å resolution were collected and processed as previously described 4 (28 047 total reflections (20-3 Å), 13 114 unique reflections (9-3 Å) used in refinement, 74% complete with R merge ) 0.062). The atomic coordinates of native rat liver arginase 4 served as the starting model for refinement with X-PLOR. 9 Refinement of the arginase-ornithine-borate complex converged smoothly to a final crystallographic R factor of 0.190 for 9-3 Å data (R free ) 0.301), with root-mean-square deviations from ideal bond lengths and angles of 0.013 Å and 1.6°, respectively.The crystal structure of the arginase-ornithine-borate complex reveals the net displacement of the manganese-bridging solvent molecule of the native enzyme by an oxygen of the tetrahedral borate anion. No other structural changes are observed in the manganese coordination polyhedra, and the...

Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.

(12) , D Morresi (12) , M Garbarino (12) , G Alberti (13) , F Valdevit (13) , E Tomelleri (14) , M Torresani (14) , G Tonon (14) , M Marchi (15) , P Corona (15) , M Marchetti (16) Forest damage inventory after the "Vaia" storm in Italy On October 29, 2018, the Vaia storm hits the NorthEastern regions of Italy by wind gusts exceeding 200 km h-1. The forests in these regions have been seriously damaged. This contribution illustrates the methodology adopted in the emergency phase to estimate forest damages caused by Vaia storm, both in terms of damaged forest areas and growing stock volume of fallen trees. 494 Municipalities registered forest damages caused by Vaia, destroyed or intensely damaged forest stands amounted to about 42