Chemical and enzymatic routes to dihydroxyacetone phosphate

Stereoselective carbon-carbon bond formation with aldolases has become an indispensable tool in preparative synthetic chemistry. In particular, the dihydroxyacetone phosphate (DHAP)-dependent aldolases are attractive because four different types are available that allow access to a complete set of diastereomers of vicinal diols from achiral aldehyde acceptors and the DHAP donor substrate. While the substrate specificity for the acceptor is rather relaxed, these enzymes show only very limited tolerance for substituting the donor. Therefore, access to DHAP is instrumental for the preparative exploitation of these enzymes, and several routes for its synthesis have become available. DHAP is unstable, so chemical synthetic routes have concentrated on producing a storable precursor that can easily be converted to DHAP immediately before its use. Enzymatic routes have concentrated on integrating the DHAP formation with upstream or downstream catalytic steps, leading to multi-enzyme arrangements with up to seven enzymes operating simultaneously. While the various chemical routes suffer from either low yields, complicated work-up, or toxic reagents or catalysts, the enzymatic routes suffer from complex product mixtures and the need to assemble multiple enzymes into one reaction scheme. Both types of routes will require further improvement to serve as a basis for a scalable route to DHA

Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered

Chemical and enzymatic routes to dihydroxyacetone phosphate

ISSN:0175-759

Exploiting Cell-Free Systems: Implementation and Debugging of a System of Biotransformations

The orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10-step catalytic system—a system of biotransformations (SBT)—for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia coli’s central carbon metabolism. This pathway consists of the endogenous glycolysis without triose-phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD+. It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7-trideoxy-D-threoheptulose-1-phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose-independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi-step enzymatic system can be readily assembled from central carbon metabolism pathways.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components