Probing the Arabidopsis Flagellin Receptor: FLS2-FLS2 Association and the Contributions of Specific Domains to Signaling Function[W][OA]

Transmembrane LRR-RLKs are a major class of plant proteins. This study investigates the functional contributions of multiple FLS2 protein domains and modifications to provide insight into structure-function relationships of LRR-RLK proteins in general

Methylated <i>N</i>^ω‑Hydroxy‑l‑arginine Analogues as Mechanistic Probes for the Second Step of the Nitric Oxide Synthase-Catalyzed Reaction

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline through the intermediate <i>N</i>^ω-hydroxy-l-arginine (NHA), producing nitric oxide, an important mammalian signaling molecule. Several disease states are associated with improper regulation of nitric oxide production, making NOS a therapeutic target. The first step of the NOS reaction has been well-characterized and is presumed to proceed through a compound I heme species, analogous to the cytochrome P450 mechanism. The second step, however, is enzymatically unprecedented and is thought to occur via a ferric peroxo heme species. To gain insight into the details of this unique second step, we report here the synthesis of NHA analogues bearing guanidinium methyl or ethyl substitutions and their investigation as either inhibitors of or alternate substrates for NOS. Radiolabeling studies reveal that <i>N</i>^ω-methoxy-l-arginine, an alternative NOS substrate, produces citrulline, nitric oxide, and methanol. On the basis of these results, we propose a mechanism for the second step of NOS catalysis in which a methylated nitric oxide species is released and is further metabolized by NOS. Crystal structures of our NHA analogues bound to nNOS have been determined, revealing the presence of an active site water molecule only in the presence of singly methylated analogues. Bulkier analogues displace this active site water molecule; a different mechanism is proposed in the absence of the water molecule. Our results provide new insights into the steric and stereochemical tolerance of the NOS active site and substrate capabilities of NOS

Enzymatic and Cryoreduction EPR Studies of the Hydroxylation of Methylated <i>N</i>^ω‑Hydroxy‑l‑arginine Analogues by Nitric Oxide Synthase from <i>Geobacillus stearothermophilus</i>

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and NO in a two-step process involving the intermediate <i>N</i>^ω-hydroxy-l-arginine (NHA). It was shown that Cpd I is the oxygenating species for l-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N^ω-OH and N^ω-H of NHA significantly inhibits the conversion of NHA into NO and l-citrulline by mammalian NOS. Kinetic studies now show that N^ω-methylation of NHA has a qualitatively similar effect on H₂O₂-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N^ω-hydroxy l-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA’s, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of l-citrulline with the intensity of the <b>g 2.26</b> EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of l-citrulline in these experiments and <i>k</i>_obs for the H₂O₂-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N^ω-OH and N^ω-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase