Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Comparative genomics of metabolic networks of free-living and parasitic eukaryotes

Background Obligate endoparasites often lack particular metabolic pathways as compared to free-living organisms. This phenomenon comprises anabolic as well as catabolic reactions. Presumably, the corresponding enzymes were lost in adaptation to parasitism. Here we compare the predicted core metabolic graphs of obligate endoparasites and non-parasites (free living organisms and facultative parasites) in order to analyze how the parasites' metabolic networks shrunk in the course of evolution. Results Core metabolic graphs comprising biochemical reactions present in the presumed ancestor of parasites and non-parasites were reconstructed from the Kyoto Encyclopedia of Genes and Genomes. While the parasites' networks had fewer nodes (metabolites) and edges (reactions), other parameters such as average connectivity, network diameter and number of isolated edges were similar in parasites and non-parasites. The parasites' networks contained a higher percentage of ATP-consuming reactions and a lower percentage of NAD-requiring reactions. Control networks, shrunk to the size of the parasites' by random deletion of edges, were scale-free but exhibited smaller diameters and more isolated edges. Conclusions The parasites' networks were smaller than those of the non-parasites regarding number of nodes or edges, but not regarding network diameters. Network integrity but not scale-freeness has acted as a selective principle during the evolutionary reduction of parasite metabolism. ATP-requiring reactions in particular have been retained in the parasites' core metabolism while NADH- or NADPH-requiring reactions were lost preferentially.

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

The glycine deportation system and its pharmacological consequences

The glycine deportation system is an essential component of glycine catabolism in man whereby 400 to 800mg glycine per day are deported into urine as hippuric acid. The molecular escort for this deportation is benzoic acid, which derives from the diet and from gut microbiota metabolism of dietary precursors. Three components of this system, involving hepatic and renal metabolism, and renal active tubular secretion help regulate systemic and central nervous system levels of glycine. When glycine levels are pathologically high, as in congenital nonketotic hyperglycinemia, the glycine deportation system can be upregulated with pharmacological doses of benzoic acid to assist in normalization of glycine homeostasis. In congenital urea cycle enzymopathies, similar activation of the glycine deportation system with benzoic acid is useful for the excretion of excess nitrogen in the form of glycine. Drugs which can substitute for benzoic acid as substrates for the glycine deportation system have adverse reactions that may involve perturbations of glycine homeostasis. The cancer chemotherapeutic agent ifosfamide has an unacceptably high incidence of encephalopathy. This would appear to arise as a result of the production of toxic aldehyde metabolites which deplete ATP production and sequester NADH in the mitochondrial matrix, thereby inhibiting the glycine deportation system and causing de novo glycine synthesis by the glycine cleavage system. We hypothesize that this would result in hyperglycinemia and encephalopathy. This understanding may lead to novel prophylactic strategies for ifosfamide encephalopathy. Thus, the glycine deportation system plays multiple key roles in physiological and neurotoxicological processes involving glycine.

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves

Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.