Functional Phenylboronate Polymers for the Recovery of Diols, Sugar Alcohols, and Saccharides from Aqueous Solution.

The ongoing transition from fossil to renewable feedstocks demands new efficient processes for an economically viable production of biomass-derived commodities and fine chemicals. Novel energy- and material-efficient product purification and separation will play a crucial role due to altered product and feed composition. The present study comprises the synthesis and tests of cross-linked p-vinylphenylboronate polymers for the separation of 18 diols, sugar alcohols, and saccharides, which can be obtained during biomass processing. The separation was based on molecular recognition, that is, esterification of the phenylboronate with vicinal diols. A correlation of the molecular complexation constant, the polymer swelling, and the maximum adsorption capacity was found. The adsorption curves over time were recorded. Preliminary results on competitive adsorption of binary mixtures showed a high potential for the separation of substrates with significantly different complexation constants. Desorption tests implied easier desorption of substrates that only adsorb on the outer polymer shell.

Structural and functional analysis of the RNA helicase Prp43 from the thermophilic eukaryote Chaetomium thermophilum.

RNA helicases are indispensable for all organisms in each domain of life and have implications in numerous cellular processes. The DEAH-box RNA helicase Prp43 is involved in pre-mRNA splicing as well as rRNA maturation. Here, the crystal structure of Chaetomium thermophilum Prp43 at 2.9 Šresolution is revealed. Furthermore, it is demonstrated that Prp43 from C. thermophilum is capable of functionally replacing its orthologue from Saccharomyces cerevisiae in spliceosomal disassembly assays.

Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry.

Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51-110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein-protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system.

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the "nineteen complex" associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.

Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22.

Splicing of pre-mRNA requires the activity of at least eight different DEAD/H-box proteins that are involved in distinct steps of the splicing process. These proteins are driving the spliceosomal machinery by ATP-dependent unwinding of dsRNA and/or disrupting protein-RNA complexes. The spliceosomal DEAH-box proteins Prp2, Prp16, Prp22 and Prp43 share homologous C-terminal domains (CTD). We have determined the crystal structure of the CTD of human Prp22 by means of MAD. The fold of the human Prp22-CTD closely resembles that of the yeast Prp43-CTD. The similarity of these helicase-associated CTDs to the winged-helix and ratchet domains of the DNA helicase Hel308 suggests an analogous function in dsRNA binding and unwinding. Here, we also demonstrate that the CTD has a significant impact on the ATPase activity of yPrp22 in vitro. Homology modeling of the CTDs of hPrp2, hPrp16 and hPrp43 suggests that the CTDs of spliceosomal helicases contain conserved positively charged patches on their surfaces representing a common RNA-binding surface as well as divergent regions most likely mediating specific interactions with different proteins of the spliceosome.

Structural insight into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase from Rhodobacter sphaeroides D.

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn(116), Ser(144), Tyr(159), and Lys(163). GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser(144), Ser(146), and Asn(151) are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.