The crystal structure of seabream antiquitin reveals the structural basis of its substrate specificity

The crystal structure of seabream antiquitin in complex with the cofactor NAD(+) was solved at 2.8A resolution. The mouth of the substrate-binding pocket is guarded by two conserved residues, Glu120 and Arg300. To test the role of these two residues, we have prepared the two mutants E120A and R300A. Our model and kinetics data suggest that antiquitin's specificity towards the substrate alpha-aminoadipic semialdehyde is contributed mainly by Glu120 which interacts with the alpha-amino group of the substrate. On the other hand, Arg300 does not have any specific interaction with the alpha-carboxylate group of the substrate, but is important in maintaining the active site conformation.     

Biosynthesis of trigonelline from nicotinate mononucleotide in mungbean seedlings

To determine the biosynthetic pathway to trigonelline, the metabolism of [carboxyl-(14)C]nicotinate mononucleotide (NaMN) and [carboxyl-(14)C]nicotinate riboside (NaR) in protein extracts and tissues of embryonic axes from germinating mungbeans (Phaseolus aureus) was investigated. In crude cell-free protein extracts, in the presence of S-adenosyl-L-methionine, radioactivity from [(14)C]NaMN was incorporated into NaR, nicotinate and trigonelline. Activities of NaMN nucleotidase, NaR nucleosidase and trigonelline synthase were also observed in the extracts. Exogenously supplied [(14)C]NaR, taken up by embryonic axes segments, was readily converted to nicotinate and trigonelline. It is concluded that the NaMN-->NaR-->nicotinate-->trigonelline pathway is operative in the embryonic axes of mungbean seedlings. This result suggests that trigonelline is synthesised not only from NAD but also via the de novo biosynthetic pathway of pyridine nucleotides.     

Soluble glucomannan isolated from Candida utilis primes blood phagocytes

It is well documented that the polysaccharide glucomannan (GM), an abundant constituent of the fungal cell wall, in the form of particulate induces strong activation of phagocytes, however, the effects of soluble GM are not known. Activation of phagocyte anti-microbial mechanisms is a crucial part of the innate host defense against invading pathogens. However, under uncontrolled inflammatory conditions they contribute to damage of surrounding tissues. Thus, to prevent these deleterious effects, the activation of phagocytes is a tightly regulated process. Therefore, in this study we analyzed the effect of soluble GM on some neutrophil functions such as reactive oxygen species production, degranulation, and receptor mobilization at the plasma membrane. Soluble GM at the tested concentrations did not stimulate oxidative burst of phagocytes directly but significantly potentiated oxidative burst in response to opsonized zymosan particles. GM induced significant phosphorylation of p47phox subunit of NADPH oxidase on Ser345. This priming effect of GM was accompanied by time and concentration dependent degranulation characterized by increased surface expression of receptors stored in neutrophil granules (CD10, CD11b, CD14, CD35, and CD66b). Degranulation was further confirmed by increase of elastase activity in media. Thus, it could be suggested that soluble GM induces priming of phagocytes connected with their degranulation, the increase of surface receptor expression, and potentiation of oxidative burst response to opsonized particles through the activation of NADPH oxidase.     

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

Potential chemoprevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from Acacia nilotica bark

Chemopreventive potential of Acacia nilotica bark extract (ANBE) against single intraperitoneal injection of N-nitrosodiethylamine (NDEA, 200 mg/kg) followed by weekly subcutaneous injections of carbon tetrachloride (CCl₄, 3 ml/kg) for 6 weeks induced hepatocellular carcinoma (HCC) in rats was studied. At 45 day after administration of NDEA, 100 and 200 mg/kg of ANBE were administered orally once daily for 10 weeks. The levels of liver injury and liver cancer markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total bilirubin level (TBL), α-feto protein (AFP) and carcinoembryonic antigen (CEA) were substantially increased following NDEA treatment. However, ANBE treatment reduced liver injury and restored liver cancer markers. ANBE also significantly prevented hepatic malondialdehyde (MDA) formation and reduced glutathione (GSH) in NDEA-treated rats which was dose dependent. Additionally, ANBE also increased the activities of antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in the liver of NDEA-administered rats. Eventually, ANBE also significantly improved body weight and prevented increase of relative liver weight due to NDEA treatment. Histological observations of liver tissues too correlated with the biochemical observations. HPLC analysis of ANBE showed the presence of gallic, protocatechuic, caffeic and ellagic acids, and also quercetin in ANBE. The results strongly support that A. nilotica bark prevents lipid peroxidation (LPO) and promote the enzymatic and non-enzymatic antioxidant defense system during NDEA-induced hepatocarcinogenesis which might be due to activities like scavenging of oxy radicals by the phytomolecules in ANBE.     

Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

Kinetic and X-ray structural evidence for negative cooperativity in substrate binding to nicotinate mononucleotide adenylyltransferase (NMAT) from Bacillus anthracis

Biosynthesis of NAD(P) in bacteria occurs either de novo or through one of the salvage pathways that converge at the point where the reaction of nicotinate mononucleotide (NaMN) with ATP is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate. This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT), which is essential for bacterial growth, making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is supported by the results of X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN- and NaAD-bound complexes were determined to resolutions of 2.50 Å, 2.60 Å and 1.75 Å, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer, supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model in which the binding affinity of substrates to the first monomer of NMAT is stronger than that to the second, and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has not been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD(P) biosynthetic pathway may occur, in part, at the reaction catalyzed by NMAT.