The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme’s preference for the 3′-phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced-fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability.     

Cox-2 gene variants in migraine

Purpose

Migraine is a multifactorial and complex disorder, and any clear diagnostic marker to assess the status of the migraineurs has not been established, yet. Nonsteroidal anti-inflammatory drugs reduce production of prostanoids including PGE2 by inhibiting COX-1 and/or COX-2, and thereby suppress inflammatory pain in patients suffering from rheumatoid arthritis, osteoarthritis, and migraine. Thus, COX-2 regulation is important in the pathogenesis and treatment of migraine. We prospectively investigated COX-2-765G → C and COX-2-1195A → G gene polymorphisms which may account for an increased risk of migraine.

Methods

The present analyses are based on 144 case subjects with migraine disease and 123 non-case subjects. Genotyping of COX-2 gene polymorphisms (COX-2-765G → C, COX-2-1195A → G) was detected by PCR-RFLP.

Results

We, for the first time, demonstrated positive association of COX-2 gene variants with an increased risk for development of migraine. Carriers of COX-2-765 C + genotype in controls were higher than in the patients (57.7% and 36.1% respectively; P < 0.0001) and the frequencies of G + genotype in patients were higher than in the controls (97.9% and 88.6% respectively; P: 0.002). In addition, frequencies of COX-2-765 GG and GC genotypes in patients were higher than in the controls (P < 0.0001, P < 0.0001 respectively). It seems that COX-2-765 G + genotype had increased and COX-2-765 C + genotype had decreased risk for migraine. In COX-2-1195 polymorphism only AG genotype was statistically significantly different in patients than in the controls (P < 0.05).

Conclusions

Our findings have suggested that COX-2-765 G + genotype could facilitate the development of migraine disease.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Leishmania amazonensis: multiple receptor-ligand interactions are involved in amastigote infection of human dendritic cells

In their mammalian hosts, Leishmania are obligate intracellular parasites that reside in macrophages and dendritic cells (DCs). In the present study, we have investigated in vitro the mechanisms of entry into human DCs of Leishmania amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein.