The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism
Thermoanaerobacterium thermosulfurigenesEM1 has been elucidated at 2.3 Å resolution. The ...final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic
R-factor of 17.9% (
R
free24.9%) with excellent stereochemistry.
The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88 – 95, 335 – 339 and 534 – 539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostability to the
T. thermosulfurigenesEM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions. The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein. Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding.
The 50% decrease in cyclization activity of the
T. thermosulfurigenesEM1 CGTase compared with that of
B. circulansstrain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88 – 95 loop. In the
T. thermosulfurigenesEM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the
B. circulansstrain 251 CGTase. The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization. Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.
Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in ...fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.
Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium ...Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.
Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets ...for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 A resolution. Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.
Alcohol oxidase, purified from the yeast
Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals ...obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 Å. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.
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