Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli.

Conservation of structure and mechanism within the transaldolase enzyme family.

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme.

Computational tools for rational protein engineering of aldolases.

In this mini-review we describe the different strategies for rational protein engineering and summarize the computational tools available. Computational tools can either be used to design focused libraries, to predict sequence-function relationships or for structure-based molecular modelling. This also includes de novo design of enzymes.

The transaldolase family: new synthetic opportunities from an ancient enzyme scaffold.

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation by aldolases offers a green, uniquely regio- and stereoselective tool with which to perform these transformations. Recent advances in the field, fueled by both discovery and protein engineering, have greatly improved the synthetic opportunities for the atom-economic asymmetric synthesis of chiral molecules with potential pharmaceutical relevance.

Broadening deoxysugar glycodiversity: natural and engineered transaldolases unlock a complementary substrate space.

The majority of prokaryotic drugs are produced in glycosylated form, with the deoxygenation level in the sugar moiety having a profound influence on the drug's bioprofile. Chemical deoxygenation is challenging due to the need for tedious protective group manipulations. For a direct biocatalytic de novo generation of deoxysugars by carboligation, with regiocontrol over deoxygenation sites determined by the choice of enzyme and aldol components, we have investigated the substrate scope of the F178Y mutant of transaldolase B, TalB(F178Y), and fructose 6-phosphate aldolase, FSA, from E.

Redesigning the active site of transaldolase TalB from Escherichia coli: new variants with improved affinity towards nonphosphorylated substrates.

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site.

Recent progress in stereoselective synthesis with aldolases.

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalysis by means of aldolases offers a unique stereoselective and green tool to perform this transformation. Recent advances in the field, fueled by either protein engineering or screening, greatly improved the number of synthetic opportunities from small chiral polyfunctional molecules to highly complex oligosaccharide analogs with potential pharmaceutical relevance.

Transaldolase: from biochemistry to human disease.

The role of the enzyme transaldolase (TAL) in central metabolism, its biochemical properties, structure, and role in human disease is reviewed. The nearly ubiquitous enzyme transaldolase is a part of the pentose phosphate pathway and transfers a dihydroxyacetone group from donor compounds (fructose 6-phosphate or sedoheptulose 7-phosphate) to aldehyde acceptor compounds. The phylogeny of transaldolases shows that five subfamilies can be distinguished, three of them with proven TAL enzyme activity, one with unclear function, and the fifth subfamily comprises transaldolase-related enzymes, the recently discovered fructose 6-phosphate aldolases.

Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin.

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg).

MJ0400 from Methanocaldococcus jannaschii exhibits fructose-1,6-bisphosphate aldolase activity.

The central carbon metabolism is well investigated in bacteria, but this is not the case for archaea. MJ0400-His(6) from Methanocaldococcus jannaschii catalyzes the cleavage of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with a V(max) of 33 mU mg(-1) and a K(m) of 430 microM at 50 degrees C. MJ0400-His(6) is inhibited competitively by erythrose-4-phosphate with a K(i) of 380 microM and displays heat stability with a half-life of c.

Microbial aldolases as C-C bonding enzymes--unknown treasures and new developments.

Aldolases are a specific group of lyases that catalyze the reversible stereoselective addition of a donor compound (nucleophile) onto an acceptor compound (electrophile). Whereas most aldolases are specific for their donor compound in the aldolization reaction, they often tolerate a wide range of aldehydes as acceptor compounds. C-C bonding by aldolases creates stereocenters in the resulting aldol products.

The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis.

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures.

Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism.

In Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G(1)/S boundary, and is transcriptionally upregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not.