In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1.34 × 104 M−1 s−1 (≈25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 106 M−1 s−1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 × 10−6 M−1 s−1 vs. 22.3 × 10−6 M−1 s−1; koff, 24.3 s−1 vs. 24.2 s−1; KO2, 0.91 × 10−6 M−1 vs. 0.92 × 10−6 M−1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 × 10−6 M−1s−1 vs. 0.51 × 10−6 M−1s−1; koff, 5.08 s−1 vs. 3.51 s−1; KCO, 1.77 × 10−7 M−1 vs. 1.45 × 10−7 M−1). All four substitutions are in the heme pocket and within 5 Å of the heme group.

Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes

Isolated subcomplexes of photosystem II from spinach (CP47RC), composed of D1, D2, cytochrome b559, CP47, and a number of hydrophobic small subunits but devoid of CP43 and the extrinsic proteins of the oxygen-evolving complex, were shown to reconstitute the Mn4Ca1Clx cluster of the water-splitting system and to evolve oxygen. The photoactivation process in CP47RC dimers proceeds by the same two-step mechanism as observed in PSII membranes and exhibits the same stoichiometry for Mn2+, but with a 10-fold lower affinity for Ca2+ and an increased susceptibility to photodamage. After the lower Ca2+ affinity and the 10-fold smaller absorption cross-section for photons in CP47 dimers is taken into account, the intrinsic rate constant for the rate-limiting calcium-dependent dark step is indistinguishable for the two systems. The monomeric form of CP47RC also showed capacity to photoactivate and catalyze water oxidation, but with lower activity than the dimeric form and increased susceptibility to photodamage. After optimization of the various parameters affecting the photoactivation process in dimeric CP47RC subcores, 18% of the complexes were functionally reconstituted and the quantum efficiency for oxygen production by reactivated centers approached 96% of that observed for reconstituted photosystem II-enriched membranes.

Genetic Dissection of Endocytic Trafficking in Drosophila Using a Horseradish Peroxidase-Bride of Sevenless Chimera: hook Is Required for Normal Maturation of Multivesicular Endosomes

Mutations in the hook gene alter intracellular trafficking of internalized ligands in Drosophila. To dissect this defect in more detail, we developed a new approach to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of HRP fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera was fully functional: it rescued the boss mutant phenotype, and its trafficking was indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss was internalized into R7 cells. In wild-type tissue, Boss accumulated in mature multivesicular bodies (MVBs) within R7 cells; such accumulation was not observed in hook eye disks, however. Quantitative electron microscopy revealed a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes were detected. Our genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Manganese oxide minerals: Crystal structures and economic and environmental significance

Manganese oxide minerals have been used for thousands of years—by the ancients for pigments and to clarify glass, and today as ores of Mn metal, catalysts, and battery material. More than 30 Mn oxide minerals occur in a wide variety of geological settings. They are major components of Mn nodules that pave huge areas of the ocean floor and bottoms of many fresh-water lakes. Mn oxide minerals are ubiquitous in soils and sediments and participate in a variety of chemical reactions that affect groundwater and bulk soil composition. Their typical occurrence as fine-grained mixtures makes it difficult to study their atomic structures and crystal chemistries. In recent years, however, investigations using transmission electron microscopy and powder x-ray and neutron diffraction methods have provided important new insights into the structures and properties of these materials. The crystal structures for todorokite and birnessite, two of the more common Mn oxide minerals in terrestrial deposits and ocean nodules, were determined by using powder x-ray diffraction data and the Rietveld refinement method. Because of the large tunnels in todorokite and related structures there is considerable interest in the use of these materials and synthetic analogues as catalysts and cation exchange agents. Birnessite-group minerals have layer structures and readily undergo oxidation reduction and cation-exchange reactions and play a major role in controlling groundwater chemistry.

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce1

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria.

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly149 to Arg) in the essential yeast gene CDC1. The mutant could be suppressed by overexpression of the SMF1 gene, which was isolated as an extragenic high-copy suppressor. The SMF1 gene codes for a highly hydrophobic protein and its deletion renders the yeast cells sensitive to low manganese concentration. In accordance with this observation, the smf1 null mutant exhibits reduced Mn2+ uptake at micromolar concentrations. Using a specific antibody, we demonstrated that Smf1p is located in the yeast plasma membrane. These results suggest that Smf1p is involved in high-affinity Mn2+ uptake. This assumption was also tested by overexpressing the SMF1 gene in the temperature-sensitive mutant of the mitochondrial processing peptidase (MAS1). SMF1 overexpression as well as addition of 1 mM Mn2+ to the growth medium complemented this mutation. This also suggests that in vivo Mas1p is a manganese-dependent peptidase. The yeast Smf1p resembles a protein from Drosophila and mammalian macrophages. The latter was implicated in conferring resistance to mycobacteria. A connection between Mn2+ transport and resistance or sensitivity to mycobacteria is discussed.