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Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide. [ABSTRACT FROM AUTHOR]

A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. [ABSTRACT FROM AUTHOR]

The response regulator VicR from the Gram-positive bacterium Enterococcus faecalis forms part of the two-component signal transduction system of the YycFG subfamily. The structure of the DNA-binding domain of VicR, VicRc, has been solved and belongs to the winged helix–turn–helix family. It is very similar to the DNA-binding domains of Escherichia coli PhoB and OmpR, despite low sequence similarity, but differs in two important loops. The α-loop, which links the two helices of the helix–turn–helix motif, is similar to that of PhoB, where it has been implicated in contacting the σ subunit of RNA polymerase, but differs from that of OmpR. Conversely, the loop following the helix–turn–helix motif is similar to that of OmpR and differs from that of PhoB. YycF/VicR, PhoB and Bacillus subtilis PhoP regulators all recognize almost identical DNA sequences and although there is currently no experimental evidence linking this loop with the DNA, the structure is consistent with possible involvement in selective DNA recognition or binding. [ABSTRACT FROM AUTHOR]

In the Gram-positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled by the transcriptional regulator AhrC. The hexameric AhrC protein binds in an L-arginine-dependent manner to pseudopalindromic operators within the promoter regions of arginine biosynthetic and catabolic gene clusters. AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription of catabolic genes. The crystal structure of AhrC has been determined at 2.7 Å resolution. Each subunit of the protein has two domains. The C-terminal domains are arranged with 32 point-group symmetry and mediate the major intersubunit interactions. The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not obey strict symmetry. A structural comparison of AhrC with the arginine repressor from the thermophile B. stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition, but also reveals some striking sequence differences, especially within the C-terminal oligomerization domain, which may contribute to the different thermostabilities of the proteins. Comparison of the crystal structure of AhrC with a 30 Å resolution model obtained by combining X-ray structure-factor amplitudes with phases derived from electron-microscopic analyses of AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more widely useful for obtaining low-resolution phase information. [ABSTRACT FROM AUTHOR]

Urease allows organisms to use exogenous and internally generated urea as a nitrogen source, by catalyzing the hydrolysis of urea to form ammonia and carbon dioxide. Urease may also participate in the systemic nitrogen-transport pathways and possibly acts as a toxic defence protein. Jack bean urease (JBU) was the first nickel- metalloenzyme identified and was crystallized as early as 1926. Despite this, the structure has not yet been determined. An antibody fragment, Fv, that has a high affinity for JBU has been used to aid crystallization. The complex, which retains full enzyme activity, forms very small crystals that diffract weakly to 3.3 Å. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 228.6, c = 130.9 Å. The structure of the urease molecule has been solved by molecular replacement using the structure of homogenous enzyme from Klebsiella aerogenes as a search model. [ABSTRACT FROM AUTHOR]