Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m 1 A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state" in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside-resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.antibiotic resistance | base flipping | RNA modification

Variants of human carbonic anhydrase II (HCA II) with amino-acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing the replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for proton transfer from His64 to the zinc-bound hydroxide in catalysis were 4 μs-1 and 9 μs-1 for Y7F and Y7F-N67Q, respectively, compared with a value of 0.8 μs-1 for wild-type HCA II. These higher values observed for Y7F and Y7F-N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with reduced branching in the networks of hydrogen-bonded water molecules between the proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5 – 1.6 Å resolution. Moreover, Y7F-N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and Nδ of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

This work examines the effect on catalysis of perturbing the position of bound CO2 in the active site of human carbonic anhydrase II (HCA II). Variants of HCA II replacing Val143 with hydrophobic residues, Ile, Leu, and Ala, were examined. The efficiency of catalysis in the hydration of CO2 for these variants was characterized by 18O exchange mass spectrometry, and their structures determined by X-ray crystallography at 1.7 to 1.5 Å resolution. The most hydrophobic substitutions V143I and V143L showed decreases in catalysis, as much as 20-fold, while the replacement by the smaller V143A showed only a moderate two-fold decrease in activity. Structural data for all three variants show no significant change in overall position of amino-acid side chains in the active site compared with wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to wild type. To further investigate the decrease in catalytic efficiency of V143I HCA II, an X-ray crystallographic CO2 entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift of the CO2 substrate towards the zinc bound solvent, placing it ~0.3 Ǻ closer than previously observed in wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the data acquisition. These data suggest that the Ile substitution at position 143 reduced catalytic efficiency is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO2 into bicarbonate and a decreased product dissociation rate.

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, β, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.

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Exploratory oxidations of several organic compounds have employed acidic solutions of vanadium(V). a-Hydroxy acids appear to undergo a stoichiometric reaction with this reagent, the products being carbon dioxide and the corresponding carboxylic acid possessing one carbon atom less than the starting substance. Ethylene glycol, glycerol, and 1,3-propanediol are oxidized to formic acid; no simple stoichiometry is observed in the vanadium(V) oxidation of malonic acid and 1,2-propanediol.

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