Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential l-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and β-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for l-Asn at 4.2 mM and kcat of 438 s−1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.

Chitin, as a fundamental polysaccharide in invertebrate skeletons, continues to be actively investigated, especially with respect to new sources and the development of effective methods for its extraction. Recent attention has been focused on marine crustaceans and sponges; however, the potential of spiders (order Araneae) as an alternative source of tubular chitin has been overlooked. In this work, we focused our attention on chitin from up to 12 cm-large Theraphosidae spiders, popularly known as tarantulas or bird-eating spiders. These organisms “lose” large quantities of cuticles during their molting cycle. Here, we present for the first time a highly effective method for the isolation of chitin from Caribena versicolor spider molt cuticle, as well as its identification and characterization using modern analytical methods. We suggest that the tube-like molt cuticle of this spider can serve as a naturally prefabricated and renewable source of tubular chitin with high potential for application in technology and biomedicine.

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A new miniprotein built from three helices, including one structure based on a ααβαααβ sequence pattern was developed. Its crystal structure revealed a compact conformation with a well-packed hydrophobic core...