Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.     

Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize ¹⁴C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS^•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS^• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS^• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP. Received: 18 February 2000 / Received revision: 24 May 2000 / Accepted: 26 May 2000     

Resolution of gastrointestinal protein loss after Helicobacter pylori eradication in a patient with hypertrophic lymphocytic gastritis

BACKGROUND: Lymphocytic gastritis is a rare condition found in approximately 1% of dyspeptic patients. An association with Helicobacter pylori infection has been described. Hypertrophic lymphocytic gastritis is a rare cause of gastrointestinal protein loss. Here, we describe a patient with hypertrophic lymphocytic gastritis, in whom gastrointestinal protein loss resolved completely following H. pylori eradication. CASE REPORT: A 38-year old obese man without gastrointestinal symptoms showed a markedly decreased serum protein (53 g/l, normal 66-85 g/l), a decreased serum albumin (33 g/l, normal 35-52 g/l) and decreased serum immunoglobulin G and immunoglobulin M levels. A renal cause for protein loss was excluded, liver function was normal. Endoscopy of the upper gastrointestinal tract revealed enlarged rigid gastric folds, and an H. pylori-associated lymphocytic gastritis. 99mTc-labelled albumin scintigraphy showed an increased activity in the upper left abdomen compatible with protein secretion in the stomach, and tracer pooling in the upper small bowel. Push enteroscopy with histology demonstrated a normal upper small bowel. Two months after eradication therapy, cure of H. pylori infection was documented and serum protein (71 g/l) and albumin (41 g/l) had returned to normal, while lymphocytic gastritis was still present. One year after eradication therapy endoscopy of the upper gastrointestinal tract and histology and laboratory values were normal. CONCLUSION: Protein-losing gastropathy caused by H. pylori-associated hypertrophic lymphocytic gastritis can be cured solely by H. pylori eradication therapy.     

Modification of ferritin during iron loading

Recombinant human ferritin loaded with iron via its own ferroxidase activity did not sediment through a sucrose-density gradient as a function of iron content. Analysis of the recombinant ferritin by native PAGE demonstrated an increase in altered migration pattern of the ferritins with increasing sedimentation, indicating an alteration of the overall charge of ferritin. Additionally, analysis of the ferritin by SDS-PAGE under nonreducing conditions demonstrated that the ferritin had formed large aggregates, which suggests disulfide bonds are involved in the aggregation. The hydroxyl radical was detected by electron spin resonance spectroscopy during iron loading into recombinant ferritin by its own ferroxidase activity. However, recombinant human ferritin loaded with iron in the presence of ceruloplasmin sedimented through a sucrose-density gradient similar to native ferritin. This ferritin was shown to sediment as a function of iron content. The addition of ceruloplasmin to the iron loading assay eliminated the detection of the DMPO-*OH adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-*OH adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water during the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed.     

A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome--deletion region at 7q11.23

Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes in a 2-cM region of chromosome band 7q11.23. With the exception of vascular stenoses due to deletion of the elastin gene, the various features of WBS have not yet been attributed to specific genes. Although >/=16 genes have been identified within the WBS deletion, completion of a physical map of the region has been difficult because of the large duplicated regions flanking the deletion. We present a physical map of the WBS deletion and flanking regions, based on assembly of a bacterial artificial chromosome/P1-derived artificial chromosome contig, analysis of high-throughput genome-sequence data, and long-range restriction mapping of genomic and cloned DNA by pulsed-field gel electrophoresis. Our map encompasses 3 Mb, including 1.6 Mb within the deletion. Two large duplicons, flanking the deletion, of >/=320 kb contain unique sequence elements from the internal border regions of the deletion, such as sequences from GTF2I (telomeric) and FKBP6 (centromeric). A third copy of this duplicon exists in inverted orientation distal to the telomeric flanking one. These duplicons show stronger sequence conservation with regard to each other than to the presumptive ancestral loci within the common deletion region. Sequence elements originating from beyond 7q11.23 are also present in these duplicons. Although the duplicons are not present in mice, the order of the single-copy genes in the conserved syntenic region of mouse chromosome 5 is inverted relative to the human map. A model is presented for a mechanism of WBS-deletion formation, based on the orientation of duplicons' components relative to each other and to the ancestral elements within the deletion region.