Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features.

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.

Physicochemical and functional properties of Cucurbita maxima pumpkin pectin and commercial citrus and apple pectins: A comparative evaluation.

The physicochemical characteristics and functional properties of pumpkin (Cucurbita maxima D. var. Cabello de Ángel) pectin obtained by cavitation facilitated extraction from pumpkin pulp have been evaluated and compared with commercial citrus and apple pectins. C. maxima pectin had an Mw value of 90 kDa and a high degree (72%) of esterification. The cytoprotective and antioxidant effects of citrus, apple and pumpkin pectin samples with different concentrations were studied in vitro in cell lines HT-29 (human colon adenocarcinoma) and MDCK1 (canine kidney epithelium). All pectin samples exhibited cytoprotective effect in HT-29 and MDCK1 cells after incubation with toxic concentrations of cadmium and mercury for 4 h. Pumpkin pectin increased the proliferation of cadmium-treated MDCK1 cells by 210%. The studied pectins also inhibited oxidative stress induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) in cell cultures, as determined by measuring the production of intracellular reactive species using dihydrochlorofluorescein diacetate (DCFH-DA). Pectin from pumpkin pomace had the highest (p < 0.05) protective effect against reactive oxygen species generation in MDCK1 cells induced by AAPH. Distinctive features of pumpkin pectin were highly branched RG-I regions, the presence of RG-II regions and the highest galacturonic acid content among the studied samples of pectins. This correlates with a considerable protective effect of C. maxima pectin against oxidative stress and cytotoxicity induced by heavy metal ions. Thus, C. maxima pectin can be considered as a source of new functional foods of agricultural origin.

Crystal structure analysis of free and substrate-bound 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans.

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.

Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization.

The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.