Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps.

The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.

Peptide-tags for enhanced DNA microarray performance.

DNA microarrays are powerful tools for gene expression analysis and genotyping studies in research and diagnostic applications. A high sensitivity and short time-to-result are prerequisites for their practical application in the clinic. The hybridization efficiency of DNA microarrays depends on the probe density and the probe orientation and thus their accessibility for target molecules. In order to find an optimal probe immobilization procedure a set of different oligonucleotide modifications was tested on epoxy silane functionalized glass slides. It was found that histidine-tagged oligonucleotides resulted in the highest amount of bound probe and by far the best hybridization efficiencies. The detection limit obtained with histidine-tagged probes was up to two orders of magnitude lower compared to commonly used probe modifications. In order to further investigate the binding mechanism of histidine-tags towards functionalized glass substrates a set of different peptide-tags with and without free terminal amino-groups and with different amino acid compositions was tested. The results indicate an impact of the terminal amino group on the covalent surface binding and of aromatic amino acid residues on the enhanced hybridisation efficiency.

6-Acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin is an activator of nitric oxide synthases.

6-Acetyl-7,7-dimethyl-7,8-dihydropterin 3 has been shown to be able to substitute for the natural cofactor of nitric oxide synthases, tetrahydrobiopterin 1, in cells and tissues that contain active nitric oxide synthases (NOSs). In both macrophages, which produce iNOS, and endothelial cells, which produce eNOS, in which tetrahydrobiopterin biosynthesis has been blocked by inhibition of GTP cyclohydrolase 1, dihydropterin 3 restored production of nitric oxide by these cells. In tissues, 3 caused relaxation in preconstricted rat aortic rings, again in which tetrahydrobiopterin biosynthesis had been inhibited, an effect that was blocked by the NOS inhibitor, L-NAME. However, dihydropterin 3 was not itself an active cofactor in purified NOS (nNOS) preparations free of tetrahydrobiopterin suggesting that intracellular reduction to 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin 4 is required for activity. Compound 4 was prepared by reduction of the corresponding 7,8-dihydropterin with sodium cyanoborohydride and has been shown to be a competent cofactor for nitric oxide production by nNOS. Together, the results show that the 7,7-dimethyl-7,8-dihydropterin is a novel structural framework for effective tetrahydrobiopterin analogues.