Efficient Rebuilding of Protein Structures

169

258

Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using ''crystal harvesting'' and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 Å, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 Å and 1.15 Å, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 Å resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.catalysis ͉ denitrification ͉ enzyme mechanism ͉ nitrite and nitric oxide binding ͉ crystal structures

Section: Methodsmentioning

confidence: 99%

Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism

Antonyuk

Strange

Sawers

et al. 2005

169

258

“…The structure was solved by molecular replacement by using the program EPMR (17) with salicylate-bound XDH (Protein Data Bank ID 1FO4) as a search model. The molecular model was built by using the program O (18). Refinement was done following standard protocols of the program CNS, Version 1.0 (19).…”

Section: Methodsmentioning

confidence: 99%

The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

Okamoto

Matsumoto²,

Hille³

et al. 2004

268

257

Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge-transfer band at Ϸ640 nm. The crystal structure of the complex indicates that the catalytically labile MoOOH oxygen has formed a bond with a carbon atom of the substrate. In addition, the MoAS group of the oxidized enzyme has become protonated to afford MoOSH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.

“…The search model for solving the structure of RasGRF1 Cdc25 was the isolated Cdc25 domain from Protein Data Bank entry 1NVV (15). Models were refined by using CNS (32) and O (33). Pymol was used for molecular illustrations (34).…”

Section: Methodsmentioning

confidence: 99%

A Ras-induced conformational switch in the Ras activator Son of sevenless

Freedman

Sondermann

Friedland

et al. 2006

123

149

The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.Cdc25 ͉ nucleotide-exchange factor ͉ crystal structure ͉ Ras exchanger motif (Rem) domain ͉ Ras guanine nucleotide-releasing factor 1