Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK.

The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK). AceK, a bifunctional enzyme, phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH) with its unique active site that harbours both the kinase and ATP/ADP-dependent phosphatase activities. AceK was the first example of prokaryotic phosphorylation identified, and the recent characterization of the structures of AceK and its complex with its protein substrate, IDH, now offers a new understanding of both previous and future endeavours.

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase.

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp).

Actin polymerization is controlled by residue size at position 204.

Previous work has shown that purified double mutant A204C/C374A yeast actin is polymerization-deficient in vitro under physiological concentrations. To understand the importance of the 204 residue in subdomain 4, a series of actin proteins with a single mutation at this position were created with Cys-374 retained. Only yeast expressing A204G-, A204S-, or A204C-actin were viable.

Overexpression of cardiac actin with baculovirus is promoter dependent.

The influence of the promoter and an N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus constructs. It was found that both non-tagged ACTC and hisACTC expression from the p10 promoter was higher than from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC.

Stealth and mimicry by deadly bacterial toxins.

Diphtheria toxin and exotoxin A are well-characterized members of the ADP-ribosyltransferase toxin family that function as virulence factors in the pathogenic bacteria Corynebacterium diphtheriae and Pseudomonas aeruginosa. Recent high-resolution structural data of the Michaelis (enzyme-substrate) complex of the P. aeruginosa toxin with an NAD(+) analog and eukaryotic elongation factor 2 (eEF2) have provided insights into the mechanism of inactivation of protein synthesis caused by these protein factors.

Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry.

The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete mono-ADP-ribosylating toxins that modify intracellular proteins. Here, we describe four structures of a catalytically active complex between a fragment of Pseudomonas aeruginosa exotoxin A (ETA) and its protein substrate, translation elongation factor 2 (eEF2). The target residue in eEF2, diphthamide (a modified histidine), spans across a cleft and faces the two phosphates and a ribose of the non-hydrolysable NAD+ analogue, betaTAD.

Characterization of oxidized nicotinamide adenine dinucleotide (NAD(+)) analogues using a high-pressure-liquid-chromatography-based NAD(+)-glycohydrolase assay and comparison with fluorescence-based measurements.

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD(+))-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD(+)) and products (adenosine 5'-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD(+) analogue, 2'-F-ribo-NAD(+), was a competing substrate or a competitive inhibitor for this toxin.

Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa.

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values.

Crystal structure of ADP-ribosylated ribosomal translocase from Saccharomyces cerevisiae.

The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 A resolution. The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor.

Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag).

Insight into the catalytic mechanism of Pseudomonas aeruginosa exotoxin A. Studies of toxin interaction with eukaryotic elongation factor-2.

The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD(+) but dependent upon pH.