Programmed Targeting Pyruvate Metabolism Therapy Amplified Single-Atom Nanozyme-Activated Pyroptosis for Immunotherapy.

Increasing cellular immunogenicity and reshaping the immune tumor microenvironment (TME) are crucial for antitumor immunotherapy. Herein, this work develops a novel single-atom nanozyme pyroptosis initiator: UK5099 and pyruvate oxidase (POx)-co-loaded Cu-NS single-atom nanozyme (Cu-NS@UK@POx), that not only trigger pyroptosis through cascade biocatalysis to boost the immunogenicity of tumor cells, but also remodel the immunosuppressive TME by targeting pyruvate metabolism. By replacing N with weakly electronegative S, the original spatial symmetry of the Cu-N4 electron distribution is changed and the enzyme-catalyzed process is effectively regulated. Compared to spatially symmetric Cu-N4 single-atom nanozymes (Cu-N4 SA), the S-doped spatially asymmetric single-atom nanozymes (Cu-NS SA) exhibit stronger oxidase activities, including peroxidase (POD), nicotinamide adenine dinucleotide (NADH) oxidase (NOx), L-cysteine oxidase (LCO), and glutathione oxidase (GSHOx), which can cause enough reactive oxygen species (ROS) storms to trigger pyroptosis. Moreover, the synergistic effect of Cu-NS SA, UK5099, and POx can target pyruvate metabolism, which not only improves the immune TME but also increases the degree of pyroptosis. This study provides a two-pronged treatment strategy that can significantly activate antitumor immunotherapy effects via ROS storms, NADH/glutathione/L-cysteine consumption, pyruvate oxidation, and lactic acid (LA)/ATP depletion, triggering pyroptosis and regulating metabolism. This work provides a broad vision for expanding antitumor immunotherapy.

The cysteine S-H stretching mode reports on long-range conformational changes in pyruvate oxidase from E. coli.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C-terminal 23 residues (the 'α-peptide'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide 'transforms' the catalytically complex EcPOX into the catalytically 'simpler' LpPOX.

Pyruvate Oxidase as a Key Determinant of Pneumococcal Viability during Transcytosis across Brain Endothelium.

Streptococcus pneumoniae invades a myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defenses governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs), formed following S. pneumoniae internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which was previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar-metabolizing enzyme that catalyzes oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of an SpxB-catalyzed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. As expected, a ΔspxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defenses to restrict microbial passage into sterile compartments. Here, by focusing on the blood-brain barrier endothelium, we investigated mechanisms that enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar-metabolizing enzyme, was found to play a crucial role in this via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide.

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Pyruvate oxidase of Streptococcus pneumoniae contributes to pneumolysin release.

BACKGROUND: Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth. RESULTS: Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis. CONCLUSIONS: Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.

Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci.

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity.

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H2O2. Streptococcus pneumoniae produces exceptionally high levels of H2O2, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H2O2 inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H2O2 production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H2O2. In vitro exposure of the spxB mutant to various H2O2 concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H2O2 or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H2O2 produced by S. pneumoniae.

Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.

In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae.

spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency approximately 3 x 10(-5)) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosidase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full virulence in a murine model of infection.

Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction.

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.

Enzymatic assay of gamma-cystathionase activity using pyruvate oxidase-peroxidase sequential reaction.

A highly sensitive method has been developed for the determination of gamma-cystathionase (EC. 4.4.1.1.) activity in rat tissues using beta-chloro-L-alanine as a substrate. This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by gamma-cystathionase, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to the gamma-cystathionase activity. The present method is more sensitive and more rapid than the usual methods and does not require troublesome steps such as centrifugation. The calibration curve is linear up to 1.6 microg of partially purified enzyme (100 U/l). Comparison with the usual method with L-homoserine as a substrate gave good correlation (r=0.990). The present method was applied to the determination of gamma-cystathionase activity in adult male rat tissues. The mean activities in liver and kidney were 8.03 and 3.91 U/g wet weight (n=10), respectively.

Prolonging cell-free protein synthesis with a novel ATP regeneration system.

A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.

Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase.

Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896-7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent, N-ethylmaleimide. Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators. An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain. These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.

2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Detection by site-specific disulfide cross-linking of a conformational change in binding of Escherichia coli pyruvate oxidase to lipid bilayers.

Escherichia coli pyruvate oxidase, a peripheral membrane homotetrameric flavoprotein, exposes its C-terminal lipid binding site in the presence of substrate pyruvate and co-factor thiamine pyrophosphate Mg2+ and binds tightly to phospholipid bilayers during catalysis. Using site-specific disulfide cross-linking, we demonstrate that disulfide cross-links are formed between C termini of D560C pyruvate oxidase and that the degree of cross-linking is greatly increased by the presence of substrate and co-factors indicating a conformational change that results in juxtaposition of two subunit C termini. The cross-linked oxidase is enzymatically active and remains able to associate with lipid micelles. These results argue strongly that lipid bilayer binding of pyruvate oxidase involves pairing of the C termini of two subunits.

An immobilized-enzyme system for the determination of sialic acid using cloned neuraminidase from Clostridium perfringens A.T.C.C. 10543.

Neuraminidase expressed by cloned nanH of Clostridium perfringens has been immobilized and employed to determine the concentration of sialic acid in human serum. Two enzyme pairs, cloned neuraminidase-N-acetylneuraminate (NANA) lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized on to 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activities of the co-immobilized neuraminidase and NANA lyase were 61 and 77%, and those of pyruvate oxidase and peroxidase were 51 and 96% of the corresponding soluble enzymes respectively. The optimal reaction pH at 37% C for each of the co-immobilized enzymes was about 1 pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of peroxidase was increased as a result of immobilization. The thermostability of the immobilized cloned neuraminidase, NANA lyase, pyruvate oxidase and peroxidase were increased 80-, 83-, 115- and 147-fold at 45 degrees C over the soluble forms respectively. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The system is thus a reliable assay method for sialic acid in serum.

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase.

BACKGROUND: The crystal structures of three thiamin diphosphate-dependent enzymes that catalyze distinct reactions in basic metabolic pathways are known. These enzymes--transketolase, pyruvate oxidase and pyruvate decarboxylase--also require metal ions such as Ca2+ and Mg2+ as cofactors and have little overall sequence similarity. Here, the crystal structures of these three enzymes are compared. RESULTS: The three enzymes share a similar pattern of binding of thiamin diphosphate and the metal ion cofactors. The enzymes function as multisubunit proteins, with each polypeptide chain folded into three alpha/beta domains. Two of these domains are involved in binding of the thiamin diphosphate and the metal ion. These domains have the same topology of six parallel beta-strands and surrounding alpha-helices. The thiamin diphosphate is bound in a cleft, formed by two domains from two different subunits. Only a few residues are conserved in all three enzymes and these are responsible for proper binding of the cofactors. CONCLUSIONS: Despite considerable differences in quaternary structure and lack of overall sequence homology, thiamin diphosphate binds to the three enzymes in a very similar fashion, and a general thiamin-binding fold can be revealed.

Minimum requirements for protease activation of flavin pyruvate oxidase.

Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.

Iron deficiency: improved exercise performance within 15 hours of iron treatment in rats.

We tested the hypothesis that a very rapid improvement in exercise performance of iron-deficient rats after treatment with iron might reveal a rate-limiting role of ionic iron as an enzyme cofactor in energy metabolism. Rats were given iron-deficient or control diets after weaning at 21 d of age and intraperitoneal iron dextran (50 mg/kg) at 45 d of age. Time to fatigue during an easy walking exercise (endurance) was measured 15 and 18 h after iron dextran or saline injection. Endurance increased more than threefold compared to the saline-treated, iron-deficient animals without a significant change in hemoglobin concentration. This prompt improvement suggests that lack of cofactor iron might play a metabolically important role in impairing exercise performance in the severely iron-deficient rat.

Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia).

By comparing environmental anaerobiosis with exercise anaerobiosis it appears that animals with high anoxia tolerance use (partly) different types of metabolic reactions to sustain energy metabolism, whereas low tolerance animals (Arthropoda, Echinodermata, Vertebrata) use the same pathway under both conditions. During exercise anaerobiosis the classical glycolysis (lactate pathway) is a main pathway among all multicellular organisms, although in marine invertebrates--except the Arthropoda and Echinodermata--it mostly does not terminate in lactate. During environmental anaerobiosis Cnidaria, Mollusca, Annelida and Sipunculida first couple additional pathways for energy extraction to the glycolytic pathway (the aspartate--succinate pathway) and later deviate the main carbon flow of glycogen at the level of phosphoenolpyruvate towards succinate, propionate and acetate production. Metabolic adaptations to anoxic cellular conditions in these groups are high fuel stores, increased ATP yield by anaerobic sources, formation of easily excretable (volatile) end products, an aspartate-dependent system for transport of hydrogen through the inner membrane of the mitochondrion and a rapid recovery from anaerobic metabolism. During anaerobic conditions three sources can contribute to the anaerobic power output, endogenous stores of both ATP and phosphagen and catabolism. Anaerobic power output rates have been calculated for a number of Mollusca, Annelida and Crustacea. Extreme anoxia resistance is coupled to a strongly reduced metabolic rate. In animals with high aspartate stores, the aspartate--succinate pathway and phosphagen hydrolysis can provide sufficient ATP during environmental anaerobiosis; however, with exercise anaerobiosis when ATP turnover rates may be increased by a factor of 20, pyruvate derivatives simultaneously accumulate in high amounts relative to succinate.