Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum. Corrigendum.

The synchrotron facilities used in collecting the data for the article by Svecová et al. [(2021), Acta Cryst. D77, 755-775] are acknowledged.

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum.

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose-methanol-choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His-Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae.

Two fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4-11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids.

Structure of laccase from Streptomyces coelicolor after soaking with potassium hexacyanoferrate and at an improved resolution of 2.3†Å.

The paper reports the structure of the small laccase from Streptomyces coelicolor determined from a crystal soaked with potassium hexacyanoferrate [K4Fe(CN)6]. The decolorization of the natively blue crystal observed upon soaking indicates the reduction of the enzyme in the crystal. The ligand binds between laccase molecules and stabilizes the crystal. The increased diffraction limit of the diffraction data collected from this crystal enabled the refinement of the small laccase structure at 2.3 Šresolution, which is the highest resolution obtained to date.

Crystallization of carbohydrate oxidase from Microdochium nivale.

Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress.

The structure of the small laccase from Streptomyces coelicolor reveals a link between laccases and nitrite reductases.

The X-ray structure of the two-domain laccase (small laccase) from Streptomyces coelicolor A3(2) was solved at 2.7-A resolution. The enzyme differs significantly from all laccases studied structurally so far. It consists of two domains and forms trimers and hence resembles the quaternary structure of nitrite reductases or ceruloplasmins more than that of large laccases. There are three trinuclear copper clusters in the enzyme localized between domains 1 and 2 of each pair of neighbor chains. In this way, a similar geometry of the active site as seen in large laccases is ensured, albeit by different arrangements of domains and protein chains. Three copper ions of type 1 lie close to one another near the surface of the central part of the trimer, and, effectively, a trimeric substrate binding site is formed in their vicinity.

Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor.

The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 A. The crystals belong to either space group P4(1)2(1)2 or P4(3)2(1)2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.