Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology.

Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-ß, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.

Corynebacterium glutamicum ArnR controls expression of nitrate reductase operon narKGHJI and nitric oxide (NO)-detoxifying enzyme gene hmp in an NO-responsive manner.

Corynebacterium glutamicum ArnR is a novel transcriptional regulator that represses expression of the nitrate reductase operon narKGHJI and the nitric oxide (NO)-detoxifying flavohemoglobin gene hmp under aerobic conditions. In a previous study, we showed that ArnR-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that ArnR senses. In this study, we show that in the absence of nitrate, ArnR-mediated repression is maintained under anaerobic conditions. The derepression in response to nitrate is eliminated by disruption of narG, suggesting that ArnR senses nitrate derivatives generated during nitrate respiration. Specifically, the hmp gene is upregulated in the presence of nitrite or nitric oxide (NO) in an ArnR-dependent manner, although the response of narK appears to be greatly affected by ArnR-independent regulation. In vitro binding of ArnR to the narK and hmp promoter regions is more strongly inhibited by NO than by nitrite. We previously showed that the UV-visible spectrum of ArnR is typical of a Fe-S cluster-containing protein. Site-directed mutagenesis of each of three cysteine residues, which are possibly involved in coordination of the cofactor in the ArnR protein, results in loss of the binding of this protein to its target promoters in vitro and eliminates the repression of the target genes in vivo under aerobic conditions. These observations suggest that the cofactor coordinated by these three cysteine residues in the ArnR protein plays a critical role in the NO-responsive expression of the narKGHJI operon and the hmp gene.

Characterization of heterologous hemoglobin and flavohemoglobin promoter regulation in Escherichia coli.

Bacterial hemoglobins and flavohemoglobins have been used to improve cell growth and productivity in biotechnological applications. The expression of globin genes can be induced by reducing the oxygen supply or applying external stressors, which provide a simple and inexpensive mechanism for induction of heterologous protein production. It is in the interest of the biotechnological industry to seek new promoters, which are non-patented, cheap and simple to induce. Therefore, new globin gene promoters have been isolated from Campylobacter jejuni, Bacillus subtilis, Deinococcus radiodurans, Streptomyces coelicolor, and Salmonella typhi. The goal was to obtain insights about the regulation mechanisms of these promoters in Escherichia coli using in silico and experimental methods. The recognition of these promoters by the E. coli transcriptional machinery was first analyzed by computational methods. Computer analysis revealed that all the promoters, except the promoter of S. coelicolor, should be functional in E. coli and most of them also contain putative binding sites for ArcA, CRP, and FNR global regulators. Furthermore, the expression profiles of the promoters fused to the chloramphenicol acetyl transferase gene were analyzed under various conditions using E. coli mutants devoid of regulatory molecules. In vivo regulation studies of globin promoters mainly verified the in silico predictions.

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution.

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.

The crystallographic structure of a human dihydropteridine reductase NADH binary complex expressed in Escherichia coli by a cDNA constructed from its rat homologue.

A human dihydropteridine reductase (EC 1.6.99.10) has been created from a rat cDNA clone by a single five-oligonucleotide mutagenesis reaction and expressed in good yield in Escherichia coli. The enzyme has been purified to homogeneity, and kinetic identity to the naturally occurring enzyme has been proven. Crystallization has also been achieved, and the crystal structure was solved using 2.5 A data that was refined to an R value of 16.9%. The structure described in this report represents the first complete structural characterization of this important human enzyme.

Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency.

Six mutations resulting in the recessive inherited disorder dihydropteridine reductase deficiency are reported, five of which are previously unknown. Two are nonsense mutations, resulting in premature termination of the protein, with the remaining four being missense mutations. The mutations found lie in the middle to 3' end of the dihydropteridine reductase reading frame, with the exception of one mutation which lies at codon 23, which is the only mutation found in more than one patient. The mutation pattern can be described as heterogeneous. The wild type and several of the mutant DHPR cDNA's were expressed in E. coli and the proteins purified and examined by a variety of techniques, including calculation of kinetic constants. One mutation (Gly23-->Asp) results in completely inactive protein, while a second (Trp108-->Gly) has substantial activity but does not completely dimerize. Both this mutant and a third, His158-->Tyr, are extremely susceptible to in vitro protease digestion, indicating that their three-dimensional structure has been altered. The protein studies underline the heterogeneous nature of DHPR mutations, in that the effects of different amino acid substitutions on the DHPR enzyme are varied.