Compound I formation is a partially rate-limiting process in chloroperoxidase-catalyzed bromination reactions

The kinetics of chloroperoxidase-catalyzed bromination and chlorination reactions were studied at various halide and hydrogen peroxide concentrations. At very high concentrations, both chloride (KI = 370 mM) and bromide (KI = 150 mM) are competitive substrate inhibitors versus hydrogen peroxide. Results at subinhibitory halide concentrations for bromination reactions (kcat = 4 ms-1, kcat/KPeroxide = 1.6 microM-1 x s-1 and kcat/KBr = 4.0 microM-1 x s-1) and chlorination reactions (kcat = 1.5 ms-1, kcat/Kperoxide = 2.3 microM-1 x s-1, and kcat/KBr = 0.32 microM-1 x s-1) indicate that halide oxidation is rate-limiting in chlorination reactions. However, in bromination reactions, both compound I formation and bromide oxidation are partially rate-limiting. This is the first documented case where compound I formation participates in determining the overall rate of a peroxidase reaction.     

Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride

Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.     

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.     

Zeta-crystallin from guinea pig lens is capable of functioning catalytically as an oxidoreductase

zeta-Crystallin, a major taxon-specific protein of the guinea pig lens, has been shown to be distantly related to the alcohol/polyol dehydrogenase family and to specifically bind NADPH. The capacity of zeta-crystallin to function catalytically was investigated in the present study. zeta-Crystallin exhibited an NADPH-dependent oxidoreductase activity with 2,6-dichlorophenolindophenol (DCIP). The NADPH:DCIP oxidoreductase activity of zeta-crystallin exhibits a linear response with increasing protein concentration, and saturation kinetics with NADPH and DCIP. This activity was abolished by heat inactivation and immunoadsorption of the protein. Dicumarol, Cibacron blue, manganese, and sulfhydryl reagents were inhibitory.     

The nuclear matrix from rat liver is capable of phosphorylating exogenous tyrosine-containing substrates

The nuclear matrix isolated from rat liver phosphorylated exogenous tyrosine-containing substrates angiotensin II and synthetic polymer (Glu, Tyr; 4:1). The phosphorylation reaction was dependent on Mn2+ or Mg2+, but the former was the preferred ion. Km values for poly(Glu,Tyr; 4:1) and ATP were 0.2 mM and 4 microM, respectively. Angiotensin II showed a lower affinity for the kinase than poly(Glu,Tyr; 4:1). The isoflavone genistein, a specific inhibitor for tyrosine phosphorylation, inhibited the tyrosine kinase activity in the nuclear matrix.     

YB-1 is capable of forming extended nanofibrils

Here we are the first to report that multifunctional Y-box binding protein 1 (YB-1) forms extended fibrils with a diameter of 15–20 nm. The YB-1 fibrils were visualized by atomic force and electron microscopy after 1-h incubation in solution with 2 M LiCl. Their length grew with incubation time and could exceed 10 μm; their shape is helical or zigzag-like. They possess polarity and tend to associate with one another to give structures of a higher order, like ribbons or bundles. The YB-1 fibrillar architecture has a distinct periodicity with a repeat unit of about 52 nm.     

Zebrafish AID is capable of deaminating methylated deoxycytidines

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

Catalase-peroxidase of Caulobacter crescentus: function and role in stationary-phase survival.

Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment. Physiological studies of oxidative and starvation stresses in C. crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase. The katG gene was shown to be solely responsible for catalase and peroxidase activity in C. crescentus. Like the katG of Escherichia coli, C. crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate. However, dramatic differences are seen in growth stage induction. E. coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase. In contrast, C. crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase. Moreover, the survival of a C. crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E. coli katE and katG null mutants. These results indicate a major role for C. crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.     

Rank-order selection is capable of maintaining all genetic polymorphisms

The fitness of organisms may be due chiefly to a fitness curve imposed on their ranking in the population with respect to heterozygosity. If this is so, then the number of polymorphisms that can be retained at a particular selective equilibrium increases as the square of the population size. All of the genetic variation that we currently observe and infer to exist can probably be maintained by selection in a population of about 10 ⁵ individuals. Selection acting in this way is so strong that these polymorphisms can be expected to behave very differently from neutral ones.     

Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin.

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.     

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.     

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.     

Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in synechocystis PCC 6803

In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co-regulation of inorganic carbon and nitrogen metabolism was established based on post-translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII-null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase-deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation.     

Human beta-defensin DEFB126 is capable of inhibiting LPS-mediated inflammation

β-Defensins are cationic, antimicrobial peptides that participate in antimicrobial defense as well as the regulation of innate and adaptive immunity. Human β-defensin 126 (DEFB126) is a multifunctional glycoprotein consisting of a conserved β-defensin core and a unique long glycosylated peptide tail. The long glycosylated peptide tail has been proven to be critical for efficient transport of sperm in the female reproductive tract, preventing their immune recognition, and efficient delivery of capacitated sperm to the site of fertilization. However, the functions of the conserved β-defensin core remain to be fully elucidated. In the present work, the conserved β-defensin core of the DEFB126 was expressed to explore its potential antimicrobial and anti-inflammatory activities. The DEFB126 core peptide exhibited both high potency for binding and neutralizing lipopolysaccharide (LPS) in vitro, and potent anti-inflammatory ability by down-regulating the mRNA expression of pro-inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in a murine macrophage cell line RAW264.7. The treatment with the DEFB126 core peptide also led to correspondingly decreased secretion of IL-6 and TNF-α. The blockade of LPS-induced p42/44 and p38 MAPK signal pathway might contribute to the anti-inflammation effects of the DEFB126 core peptide. Furthermore, fluorescence-labeled DEFB126 could enter RAW 264.7 cells and reduce the production of LPS-stimulated inflammatory factors, implying that DEFB126 might also participate in intracellular regulation beyond its direct LPS neutralization. In summary, our results demonstrate that the DEFB 126 core peptide has critical functions in parallel to its C-terminal tail by showing LPS-binding activity, anti-inflammatory effects and intracellular regulatory function.     

Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) beta-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl beta-D-glucuronide in contrast to bovine liver beta-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of beta-glucuronidase. A number of natural beta-glucuronides were screened by competitive inhibition for KLFc beta-glucuronidase. We found that steroid beta-glucuronides such as beta-estradiol 3-beta-D-glucuronide, estrone 3-beta-D-glucuronide, and estriol 3beta-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel beta-glucuronidase and that steroid beta-glucuronides are potential candidates for the natural substrate(s) of Klotho.     

Catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803: cloning, overexpression in Escherichia coli, and kinetic characterization.

The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pl = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent Km = 4.9 +/- 0.25 mM and apparent kcat = 3500 s(-1)) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 +/- 0.26) x 10(3) M(-1) s(-1), whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 +/- 0.03) x 10(6) M(-1) S(-1), (8.62 +/- 0.21) x 10(4) M(-1) S(-1), and (5.43 +/- 0.19) x 10(3) M(-1) S(-1), respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 +/- 0.1) x 10(5) M(-1) S(-1) was obtained.