Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from corynebacterium diphtheriae: x-ray crystallography and molecular dynamics investigation.

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

Characterization of C-S Lyase from C. diphtheriae: a possible target for new antimicrobial drugs.

The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5'-phosphate (PLP-) dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics.

[The ultrastructural localization of NO-synthase NADPH diaphorase in a peripheral nerve and its change in diphtheritic polyneuropathy]. / Ul'trastrukturnaia lokalizatsiia NO-sintaznoi NADPH-diaforazy v perifericheskom nerve i ee izmenenie pri difteriinoi polineiropatii.

Light and electron microscopy was used to study the distribution and changes of NADPH-diaphorase in the cutaneous nerve biopsy specimens in different periods of diphtheritic polyneuropathy (DP). there was a reduction in the reaction rate of the enzyme in Schwann's cells of the destructively changed nerve fibers and an increase in the remyelinated nerve fibers. The enzyme is located on the nuclear and endoplasmic reticulum membranes and ribosomes. It is suggested that there is an association of the synthesis of nitric oxide with the myelin-producing function of Schwann's cells.

[Isoenzymes of transferases in serum from diphtheria patients]. / Izofermenty transferaz v syvorotke krovi bol'nykh difteriei.

AIM: Investigation of serum spectrum of isoenzymes AlAT, AsAT, GGT in diphtheria in adults. MATERIALS AND METHODS: The serum spectrum of cytoplasmic and mitochondrial isoenzymes AlAT, AsAT, GGT were studied in 12 bacterium carriers and 65 patients with diphtheria. Quite original evidence has been obtained on mechanism of hyperenzymemia. RESULTS: Diphtheria is associated with enhanced activity of cytoplasmic isoenzymes in correlation with intoxication severity. Spectrum of isoenzymes AlAT and especially GGT shifted to the side of mitochondrial fractions. There were no statistically significant differences in the activity and spectrum of isoenzymes in myocarditis and in its absence. In clinical recovery the spectrum of isoenzyme activity remained changed. CONCLUSION: It is suggested that dysisoenzymemia is related to active role of hepatocytes in utilization of diphtheria toxin, that carriers are not healthy people as hyperenzymemia is associated with morphological changes in the myocardium.

Bull-neck, electrocardiographic changes and creatine phosphokinase blood levels in patients with diphtheria.

All paediatric patients with the diagnosis of diphtheria who were seen at the Department of Child Health Ujung Pandang General Hospital from October 1987 to October 1989 were evaluated for bull-neck, ECG patterns and serum creatine phosphokinase (CPK) values. Their ages ranged from 1 year and 2 months to 13 years with a mean of 6.54 +/- 3.09 years. Males and females were affected in a ratio of 1:1. Of 39 patients included in the trial, 28 were classified as having normal ECG and 11 as having abnormal ECG on admission. During hospitalization, 56.4% of cases showed ECG changes. All patients had raised serum CPK levels on admission. This increase was 14.6 times the normal level in cases with abnormal ECG and only 3.0 times in those with normal ECG (p < 0.01). The highest levels of serum CPK were noted in the first week, then returned to normal in the second week and decreased further in the third week. It became also evident that patients who developed abnormal ECG later on, had already demonstrated an increased serum CPK level of 5.2 times the normal levels on admission. Bull-neck appeared in 19 out of the 39 patients. Patients with bull-neck differed very significantly (p < 0.001) from those without bull-neck in either the frequency of the occurrence of abnormal ECG patterns or the mean CPK serum levels.(ABSTRACT TRUNCATED AT 250 WORDS)

[Lymphocyte and neutrophil cytochemistry in the dynamics of different forms of diphtheria in adults]. / Tsitokhimiia limfotsitov i neitrofilov v dinamike razlichnykh form difterii u vzroslykh.

The cytochemical study of lymphocytes and neutrophils isolated from fractionated blood of diphtheria patients and carriers has revealed that a decrease in the activity of lymphocyte succinic dehydrogenase and myeloperoxidase can be observed at all periods of the disease and in all its forms. A decrease in the activity of lymphocyte nonspecific esterase has been noted only in patients with toxic and subtoxic diphtheria and a decrease in the activity of neutrophil alkaline phosphatase, in carriers. The analysis of correlations between the parameters of five enzymes under study (lymphocyte succinic dehydrogenase, lymphocyte acid phosphatase, lymphocyte non-specific esterase, myeloperoxidase, neutrophil alkaline phosphatase) and enzymatic rosette parameters has been made. The analysis has revealed an essential increase in the number of correlations in comparison with donors, changes in the qualitative nature of these correlations and sometimes the reversion of the correlations. Carriers have shown the greatest number of correlations. By the end of the terms of observations no restoration of normal correlations has been observed.

[Protective lysosomal autoregulatory mechanism and its disorders in diphtheric intoxication]. / Zashchitnyi lizosomnyi samoreguliatornyi mekhanizm i ego narushenie pri difteriinoi intoksikatsii.

Diphterial intoxication was established in rabbits by means of bolus 1 DLM/kg intravenous injection of diphterial toxin. Different types of N-acetyl-beta-1)-galactosaminidase's activity were determined in lysosomal fraction which had been obtained after ultracentrifugation of the left and right ventricles' tissue of the heart. It has been found that the mechanism of fixation's reinforcement of this enzyme exists in lysosomes in case of increasing of its activity. This mechanism was damaged on the different stages of diphterial intoxication that influenced negatively the heart's contractility.

[Changes in the activity of lysosomal enzymes and in the indices of cardiac contractile function in experimental diphtheria poisoning in rabbits]. / Izmenenie aktivnosti fermentov lizosom i nekotorykh pokazatelei sokratitel'noi funktsii serdtsa pri éksperimental'noi difteriinoi intoksikatsii u krolikov.

Contractility of the heart ventricles and the activity of N-acetyl-beta-D-galactosaminiidase in fraction of lysosomes of this part of the heart were studied in rabbits with experimental diphtheritic intoxication. Three days after the onset of the process changes in the activity of the enzyme reached maximum and were expressed in increase of values indicating increased transfer of enzymes from the particles into the cytosol. At the same time, values which were evidence of fixation of the enzyme in the lysosomes reduced. Correlation analysis showed that intensified escape of enzymes into the cytoplasm led to weakening of heart contractile function.

Alkaline phosphatase in the thymus.

(1) Fetal thymuses, organs from patients who died from diseases that are not clinically known to be associated with concomitant lymphoid tissue involvement, as well as thymuses from patients dying from diseases which effect the lymphatic complex of the body, one way or another, have been investigated for their alkaline phosphatase activity, using Gomori technique and applying four different phosphate esters as substrates. (2) Three substrates (beta-glycerophophate, riboflavin 5-phosphate and adenosine triphosphate) showed essentially the same pattern of activity in which the cortex and Hassall's corpuscles were reactive, while the medulla was negative. A reversal of this pattern was demonstrated with 5-monophosphoric acid. (3) Before the age of 32-36 weeks of intra-uterine life there is no alkaline phosphatase activity in the thymus; therafter, the enzyme begins to make its first appearance. (4) There is a definite increase in the intensity of the reaction with advance of intra-uterine life. This increase in phosphatase content is continued postnatally, to reach its maximum at about the age of 10 years: after that, the enzyme activity gradually subsides. (5) There is a tremendous augmentation of phosphatase activity in the case of disease which are known to affect the lymphoid complex. (6) The phosphatase activity of the thymus has been discussed in relation to the prevailing concepts about the function of the thymus, with special emphasis on a possible association with 'lymphocyte-stimulating factor' production and/or secretion.