Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme delta9 stearoyl-acyl carrier protein desaturase. Implications for oxygen activation and catalytic intermediates

Delta9 stearoyl-acyl carrier protein (ACP) desaturase is a mu-oxo-bridged di-iron enzyme, which belongs to the structural class I of large helix bundle proteins and that catalyzes the NADPH and O2-dependent formation of a cis-double bond in stearoyl-ACP. The crystal structures of complexes with azide and acetate, respectively, as well as the apoand single-iron forms of Delta9 stearoyl-ACP desaturase from Ricinus communis have been determined. In the azide complex, the ligand forms a mu-1,3-bridge between the two iron ions in the active site, replacing a loosely bound water molecule. The structure of the acetate complex is similar, with acetate bridging the di-iron center in the same orientation with respect to the di-iron center. However, in this complex, the iron ligand Glu196 has changed its coordination mode from bidentate to monodentate, the first crystallographic observation of a carboxylate shift in Delta9 stearoyl-ACP desaturase. The two complexes are proposed to mimic a mu-1,2 peroxo intermediate present during catalytic turnover. There are striking structural similarities between the di-iron center in the Delta9 stearoyl-ACP desaturase-azide complex and in the reduced rubrerythrin-azide complex. This suggests that Delta9 stearoyl-ACP desaturase might catalyze the formation of water from exogenous hydrogen peroxide at a low rate. From the similarity in iron center structure, we propose that the mu-oxo-bridge in oxidized desaturase is bound to the di-iron center as in rubrerythrin and not as reported for the R2 subunit of ribonucleotide reductase and the hydroxylase subunit of methane monooxygenase. The crystal structure of the one-iron depleted desaturase species demonstrates that the affinities for the two iron ions comprising the di-iron center are not equivalent, Fe1 being the higher affinity site and Fe2 being the lower affinity site.     

Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.     

Origin of the Hawaiian endemic mints within North American Stachys (Lamiaceae)

The Hawaiian endemic mints constitute a major island radiation, displaying a remarkable diversity of floral, fruit, and vegetative features. Haplostachys and Phyllostegia have flowers associated with insect pollination, whereas Stenogyne has flowers typical of bird pollination. The three genera had been thought to be closely related to East Asian members of Lamioideae tribe Prasieae because of the fleshy nutlets borne by Phyllostegia and Stenogyne. We evaluated the origins of the Hawaiian mints using phylogenetic analyses of DNA sequence data from the plastid rbcL and trnL intron loci and the nuclear ribosomal 5S nontranscribed spacer. The Hawaiian genera were found to be monophyletic but deeply nested inside another lamioid genus, Stachys. In particular, they were found to be most closely related to a group of temperate North American Stachys from the Pacific coast, suggesting that the Hawaiian mints derived from a single colonization event from western North America to the Hawaiian Islands. Furthermore, Stachys, which contains amphiatlantic and transberingian clades, was found to be polyphyletic, with some species more closely related to Gomphostemma, Phlomidoschema, Prasium, and Sideritis than to other species of Stachys. Based on chromosomal evidence and our phylogenetic analyses, we hypothesize that the Hawaiian mints may be polyploid hybrids whose reticulate genomes predate the Hawaiian dispersal event and are derived from Stachys lineages with flowers exhibiting insect- vs. bird-pollination characteristics. Thus, the Hawaiian endemic mints may provide yet another insular system for the combined study of polyploidy, hybrid cladogenesis, and adaptive radiation.     

Overlapping and specific patterns of GDNF,c-ret and GFR alpha mRNA expression in the developing chicken retina

GDNF and the GDNF receptors, c-Ret, GFR alpha 1 and 2 mRNA is expressed in the developing chicken retina. GDNF labelling was mainly found in embryonic day 4-5 retina but weak labelling could also be found over scattered retinal cells at later stages. c-ret labelling was found over ganglion cells, amacrine and horizontal cells; the preferred GDNF receptor (GFR alpha 1) over amacrine and horizontal cells; and the less preferred GDNF receptor (GFR alpha 2) over ganglion cells, amacrine cells and photoreceptors.     

Expression of the AMBP gene transcript and its two protein products,alpha(1)-microglobulin and bikunin,in mouse embryogenesis

The expression pattern of the alpha(1)-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5-15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The alpha(1)-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: alpha(1)-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins.     

Studies of the mechanism of phenol hydroxylase: effect of mutation of proline 364 to serine

An active site residue in phenol hydroxylase (PHHY), Pro364, was mutated to serine to investigate its role in enzymatic catalysis. In the presence of phenol, the reaction between the reduced flavin of P364S and oxygen is very fast, but only 13% of the flavin is utilized to hydroxylate the substrate, compared to nearly 100% for the wild-type enzyme. The oxidative half-reaction of PHHY using m-cresol as a substrate is similarly affected by the mutation. Pro364 was suggested to be important in stabilizing the transition state of the oxygen transfer step by forming a hydrogen bond between its carbonyl oxygen and the C4a-hydroperoxyflavin [Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. The P364S mutation may weaken this interaction by increasing the flexibility of the peptide chain; hence, the transition state would be destabilized to result in a decreased level of hydroxylation of phenol. However, when the oxidative half-reaction was studied using resorcinol as a substrate, the P364S mutant form was not significantly different from the wild-type enzyme. The rate constants for all the reaction steps as well as the hydroxylation efficiency (coupling between NADPH oxidation and resorcinol consumption) are comparable to those of the wild-type enzyme. It is suggested that the function of Pro364 in catalysis, stabilization of the transition state, is not as important in the reaction with resorcinol, possibly because the position of hydroxylation is different with resorcinol than with phenol and m-cresol.     

Microcephaly, microphthalmia, congenital cataract, with calcification of the basal ganglia: MCA/MR syndrome

We present a Hungarian girl with microcephaly, microphthalmia, congenital cataract, prominent nasal root, peaked nose, micrognathia with high arched palate, mild mental retardation, calcification of the basal ganglia and serology for the connatal infections. We suggest that our proband may be an allelic variant of COFS syndrome.     

Protection against cigarette smoke-induced damage to intact transformed rabbit corneal cells by N-acetyl-L-cysteine

In order to assess cigarette smoke-induced oxidative damage to intact cells, an assay was developed to measure cell detachment and protection. Due to the complex nature of cigarette smoke, which contains molecules that can interfere with conventional spectrophotometric and fluorometric biochemical assays, transformed rabbit corneal cells were radiolabeled with tritiated thymidine and then subjected to direct stream smoke. As a result, cell damage in response to the smoke from only two cigarettes could be measured in a time-dependent manner. When cells were prelabeled with N-acetyl-L-cysteine (NAC), a substrate for glutathione synthesis, a significant reduction in damage was measured. Additionally, when buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, was incubated with cells, a reduction in the effectiveness of NAC was observed, although NAC still retained some activity. Furthermore, vitamin E conferred no protection to cells in this system nor was NAC active in a separate assay that appears to favor peroxyl radical generation. From these results we conclude that cigarette smoke damage can easily be determined at the cellular level with this technique and that NAC acted to prevent this damage in two ways: first, as glutathione precursor and, secondly, as an antioxidant capable of scavenging non-peroxyl radicals.