The Conversion of UDP-Glc to UDP-Man: In Silico and Biochemical Exploration To Improve the Catalytic Efficiency of CDP-Tyvelose C2-Epimerases.

A promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus (TaTyvE) belonging to the nucleotide sugar active short-chain dehydrogenase/reductase superfamily (NS-SDRs) was recently discovered. TaTyvE performs the slow conversion of NDP-glucose (NDP-Glc) to NDP-mannose (NDP-Man). Here, we present the sequence fingerprints that are indicative of the conversion of UDP-Glc to UDP-Man in TyvE-like enzymes based on the heptagonal box motifs. Our data-mining approach led to the identification of 11 additional TyvE-like enzymes for the conversion of UDP-Glc to UDP-Man. We characterized the top two wild-type candidates, which show a 15- and 20-fold improved catalytic efficiency, respectively, on UDP-Glc compared to TaTyvE. In addition, we present a quadruple variant of one of the identified enzymes with a 70-fold improved catalytic efficiency on UDP-Glc compared to TaTyvE. These findings could help the design of new nucleotide production pathways starting from a cheap sugar substrate like glucose or sucrose.

A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases.

Nucleotide sugar 4,6-dehydratases belong to the Short-chain Dehydrogenase/Reductase (SDR) superfamily and catalyze the conversion of an NDP-hexose to an NDP-4-keto-6-deoxy hexose, a key step in the biosynthesis of a plethora of deoxy and amino sugars. Here, we present a colorimetric assay for the detection of their reaction products (NDP-4-keto-6-deoxy hexoses) using concentrated sulfuric acid and an ethanolic resorcinol solution. Under these conditions, the keto-function of the dehydratase product reacts specifically with resorcinol to form an orange-red or pink complex for NDP-glucose/GDP-mannose and UDP-N-acetylglucosamine, respectively, with an absorption maximum at 510 nm. The presented assay allows reliable product detection at low concentrations and can be applied in microtiter plates. It thus allows the determination of kinetic enzyme parameters like the optimal temperature, pH, Vmax, KM and kcat, as well as the miniaturization for screening purposes with crude cell extracts. As such, this detection assay opens new possibilities for the characterization and screening of these dehydratases in 96-well plates for different research goals.

Nucleotide sugar dehydratases: Structure, mechanism, substrate specificity, and application potential.

Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies.

Source distribution of neuromagnetic slow wave activity in schizophrenic and depressive patients.

OBJECTIVE: Focal slow waves in the delta and theta frequency range frequently appear in psychopathological conditions. Due to their focal nature they can be localized by dipole modeling. We previously reported regional clustering of slow waves in temporal and parietal cortex of schizophrenic patients whereas such activity is largely absent in normals. Here we examine, to what extent distribution of slow wave generators differentiates schizophrenic from depressive syndromes. METHODS: The regional densities of generators of focal slow waves were determined during resting conditions in patients with DSM-IV diagnoses of schizophrenia (N=25) and depression (N=27) and in 18 healthy controls. RESULTS: Schizophrenic patients demonstrated accentuated temporal and parietal delta and theta dipole clustering, when compared to both the control and the depressive sample. In contrast, depressive patients had reduced frontal and prefrontal delta and theta dipole density relative to both schizophrenics and controls. This pattern was not related to age. Men generally displayed somewhat higher slow wave activity than women. For the areas of most pronounced slow wave deviances activity within each group was related to symptom scores: higher left-temporal slow wave activity was associated with hallucinations in schizophrenics, suppression of left-prefrontal slow wave activity correlated with depression scores. CONCLUSIONS: Results suggest that slow wave distribution may assist in differentially diagnosing psychopathological conditions.