A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.

The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.     

The crystal structure of exfoliative toxin B: a superantigen with enzymatic activity.

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.     

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influences enzymatic activity

UGPase (UDP-glucose pyrophosphorylase) is highly conserved among eukaryotes. UGPase reversibly catalyses the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites, and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. In the present study, the crystal structure of hUGPase (human UGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase differs distinctly from yUGPase (yeast UGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly affect the UGPase activity. Moreover, this latch effect is also evolutionarily meaningful in UGPase from different species.     

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.     

Gastric lipase: crystal structure and activity

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.     

Cysteine conjugate S-oxidase. Characterization of a novel enzymatic activity in rat hepatic and renal microsomes

Cysteine conjugate S-oxidase activity, with S-benzyl-L-cysteine as substrate, was found mostly in the microsomal fractions of rat liver and kidney. In the presence of oxygen and NADPH, S-benzyl-L-cysteine is converted to S-benzyl-L-cysteine sulfoxide; no S-benzyl-L-cysteine sulfone was detected. The Vmax for S-benzyl-L-cysteine sulfoxide formation by kidney microsomes was nearly 3-fold greater than the rate measured with liver microsomes. Inclusion of catalase, superoxide dismutase, glutathione, butylated hydroxyanisole, the peroxidase inhibitor, potassium cyanide, the cytochrome P-450 inhibitors, 1-benzylimidazole and metyrapone, or a monoclonal antibody to cytochrome P-450 reductase did not inhibit the metabolic reaction. Flavin-containing monooxygenase alternate substrates, N,N-dimethylaniline, n-octylamine, and methimazole inhibited the S-oxidase activities. Analogues of S-benzyl-L-cysteine, S-methyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine inhibited the S-benzyl-L-cysteine S-oxidase activities, whereas S-carboxymethyl-L-cysteine and S-benzyl-L-cysteine methyl ester had no effect. These results provide clear evidence against the involvement of reactive oxygen intermediates or cytochrome P-450 in the sulfoxidation of S-benzyl-L-cysteine and indicate that the S-oxidase activities may be associated with flavin-containing monooxygenases which exhibit selectivity in the interaction with cysteine S-conjugates.     

Protein tyrosine phosphatase: enzymatic assays

Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.     

The crystal structure,mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad

Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.     

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.     

The tetrameric structure of Plasmodium falciparum phosphoglycerate mutase is critical for optimal enzymatic activity

The glycolytic enzyme phosphoglycerate mutase (PGM) is of utmost importance for overall cellular metabolism and has emerged as a novel therapeutic target in cancer cells. This enzyme is also conserved in the rapidly proliferating malarial parasite Plasmodium falciparum, which have a similar metabolic framework as cancer cells and rely on glycolysis as the sole energy-yielding process during intraerythrocytic development. There is no redundancy among the annotated PGM enzymes in Plasmodium, and PfPGM1 is absolutely required for the parasite survival as evidenced by conditional knockdown in our study. A detailed comparison of PfPGM1 with its counterparts followed by in-depth structure-function analysis revealed unique attributes of this parasitic protein. Here, we report for the first time the importance of oligomerization for the optimal functioning of the enzyme in vivo, as earlier studies in eukaryotes only focused on the effects in vitro. We show that single point mutation of the amino acid residue W68 led to complete loss of tetramerization and diminished catalytic activity in vitro. Additionally, ectopic expression of the WT PfPGM1 protein enhanced parasite growth, whereas the monomeric form of PfPGM1 failed to provide growth advantage. Furthermore, mutation of the evolutionarily conserved residue K100 led to a drastic reduction in enzymatic activity. The indispensable nature of this parasite enzyme highlights the potential of PfPGM1 as a therapeutic target against malaria, and targeting the interfacial residues critical for oligomerization can serve as a focal point for promising drug development strategies that may not be restricted to malaria only.     

The GID ubiquitin ligase complex is a regulator of AMPK activity and organismal lifespan

ABSTRACT

The AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by sensing the metabolic status of the cell. AMPK is regulated by phosphorylation and dephosphorylation as a result of changing AMP/ATP levels and by removal of inhibitory ubiquitin residues by USP10. In this context, we identified the GID-complex, an evolutionarily conserved ubiquitin-ligase-complex (E3), as a negative regulator of AMPK activity. Our data show that the GID-complex targets AMPK for ubiquitination thereby altering its activity. Cells depleted of GID-subunits mimic a state of starvation as shown by increased AMPK activity and macroautophagic/autophagic flux as well as reduced MTOR activation. Consistently, gid-genes knockdown in C. elegans results in increased organismal lifespan. This study may contribute to understand metabolic disorders such as type 2 diabetes mellitus and morbid obesity and implements alternative therapeutic approaches to alter AMPK activity.     

Induced structure of a helical switch as a mechanism to regulate enzymatic activity

Herpesviruses encode a protease that is activated by homodimerization at high enzyme concentrations during lytic replication. The homodimer contains two active sites, which are distal from the dimer interface. Assignment of backbone NMR resonances and engineering of a redox switch show that two helices position a loop containing catalytic residues within each active site.     

Vaccinia virus encodes a polypeptide with DNA ligase activity.

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.     

POSH2 is a RING finger E3 ligase with Rac1 binding activity through a partial CRIB domain

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.

Structured summary

MINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)     

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.     

X-ray crystal structure and antioxidant activity of salidroside, a phenylethanoid glycoside

The X-ray single-crystal structure of natural salidroside (=2-(4-hydroxyphenyl)ethyl beta-D-glucopyranoside; 1), isolated from Cistanche deserticola, is reported for the first time, as well as its absolute configuration. The radical-scavenging activity of 1 towards the superoxide radical anion (O*2-) was determined experimentally by chemiluminescence measurements of the pyrogallol-luminol system, and compared to that of the corresponding aglycone, i.e., tyrosol (=4-(2-hydroxyethyl)phenol; 2).