Characterization of a new AAA+ protein from archaea

We investigated a new archaeal member of the AAA+ protein family (ATPases associated with various cellular activities) which is found in all methanogenic archaea and the sulphate-reducer Archaeoglobus fulgidus. These proteins cluster to COG1223 predicted to form a subgroup of the AAA+ ATPases. The gene from A. fulgidus codes for a protein of 40 kDa monomeric molecular weight, which we overexpressed in Escherichia coli and purified to homogeneity. The protein forms ring-shaped complexes with a diameter of 125A as determined by electron microscopy. Using sedimentation equilibrium analysis we demonstrate that it assembles into hexamers over a wide concentration range both in presence and absence of ATP. As suggested by homology to other members of the AAA+ family, the complex binds and hydrolyzes ATP. Michaelis-Menten analysis revealed a k(cat) of 118 min(-1) and a K(M) of 1.4 mM at 78 degrees C. This hyperthermophilic archaeal ATPase is stable to 86 degrees C and the ATPase activity is maximal at this temperature. The protein is most homologous to the AAA-domain of FtsH from bacteria, while the N-terminal domain shows predicted structural homology to members of the CDC48 family of AAA proteins. Possible roles of this new AAA+ protein are discussed.     

A 200-amino acid ATPase module in search of a basic function

A fast growing family of ATPases has recently been highlighted. It was named the AAA family, for ATPases Associated to a variety of cellular Activities. The key feature of the family is a highly conserved module of 230 amino acids present in one or two copies in each protein. Despite extensive sequence conservation, the members of the family fulfil a large diversity of cellular functions: cell cycle regulation, gene expression in yeast and HIV, vesicle-mediated transport, peroxisome assembly, 26S protease function etc. In addition, several members of this family can be found in the same organism (up to 17 in S. cerevisiae). The contrast between functional diversity and structural conservation of the module, from archaebacteria to mammals, suggests that it plays an essential, but as yet unknown, role at key points of the cellular machinery. Two (non-exclusive) such possibilities are: (1) ATP-dependent proteasome function and (2) ATP-dependent anchorage of proteins. Finally, the basic biochemical activity of the AAA module is still a matter of speculation, and we propose that it acts as an ATP-dependent protein clamp.     

The torsin-family AAA+ protein OOC-5 contains a critical disulfide adjacent to Sensor-II that couples redox state to nucleotide binding

A subgroup of the AAA+ proteins that reside in the endoplasmic reticulum and the nuclear envelope including human torsinA, a protein mutated in hereditary dystonia, is called the torsin family of AAA+ proteins. A multiple-sequence alignment of this family with Hsp100 proteins of known structure reveals a conserved cysteine in the C-terminus of torsin proteins within the Sensor-II motif. A structural model predicts this cysteine to be a part of an intramolecular disulfide bond, suggesting that it may function as a redox sensor to regulate ATPase activity. In vitro experiments with OOC-5, a torsinA homolog from Caenorhabditis elegans, demonstrate that redox changes that reduce this disulfide bond affect the binding of ATP and ADP and cause an attendant local conformational change detected by limited proteolysis. Transgenic worms expressing an ooc-5 gene with cysteine-to-serine mutations that disrupt the disulfide bond have a very low embryo hatch rate compared with wild-type controls, indicating these two cysteines are essential for OOC-5 function. We propose that the Sensor-II in torsin family proteins is a redox-regulated sensor. This regulatory mechanism may be central to the function of OOC-5 and human torsinA.     

Biochemical coupling of the two nucleotide binding domains of ClpB: covalent linkage is not a prerequisite for chaperone activity

ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle.     

The Pseudomonas aeruginosa devB/SOL homolog, pgl, is a member of the hex regulon and encodes 6-phosphogluconolactonase

A cyclic version of the Entner-Doudoroff pathway is used by Pseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the hex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOL family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5' end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgl and propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.     

Cloning, sequencing, and characterization of the gene encoding FrpB, a major iron-regulated, outer membrane protein of Neisseria gonorrhoeae.

FrpB (for Fe-regulated protein B) is a 76-kDa outer membrane protein that is part of the iron regulon of Neisseria gonorrhoeae and Neisseria meningitidis. The frpB gene from gonococcal strain FA19 was cloned and sequenced. FrpB was homologous to several TonB-dependent outer membrane receptors of Escherichia coli as well as HemR of Yersinia enterocolitica and CopB of Moraxella catarrhalis. An omga insertion into the frpB coding sequence caused a 60% reduction in 55Fe uptake from heme, but careful analysis suggested that this effect was nonspecific. While FrpB was related to the family of TonB-dependent proteins, a function in iron uptake could not be documented.     

The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor.

In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases. We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris. Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD. Consistent with this observation, CG4 patients carry mutations in PXAAA1. The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein. Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase. Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p. We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity.     

Use of a phoB'-'lacZ fusion gene to determine the N-terminal amino acid sequence of the phoB protein and to prepare antiserum against the protein

phoB is a positive regulatory gene of the phosphate regulon of Escherichia coli. A plasmid carrying a phoB'-'lacZ fusion gene was constructed by in vitro recombination. A PhoB-LacZ hybrid protein was purified from the cells carrying the plasmid by monitoring beta-galactosidase activity. The amino-terminal amino acid sequence of the PhoB protein was determined by utilizing the hybrid protein. Antiserum against the PhoB protein was prepared by immunizing rabbits with the hybrid protein. The serum thus prepared showed specificity for both the PhoB protein and beta-galactosidase.     

Involvement of <Emphasis Type="Italic">soxRS</Emphasis> Regulon in Response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Oxidative Stress Induced by Hydrogen Peroxide

The effect of hydrogen peroxide on the activity of soxRS and oxyR regulon enzymes in different strains of Escherichia coli has been studied. Treatment of bacteria with 20 μM H₂O₂ caused an increase in catalase and peroxidase activities (oxyR regulon) in all strains investigated. It is shown for the first time that oxidative stress induced by hydrogen peroxide causes in some E. coli strains a small increase in activity of superoxide dismutase and glucose-6-phosphate dehydrogenase (soxRS regulon). This effect is cancelled by chloramphenicol, an inhibitor of protein synthesis in prokaryotes. The increase in soxRS regulon enzyme activities was not found in the strain lacking the soxR gene. These results provide evidence for the involvement of the soxRS regulon in the adaptive response of E. coli to oxidative stress induced by hydrogen peroxide. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1506–1513. Original Russian Text Copyright ? 2005 by Semchyshyn, Bagnyukova, Lushchak.