Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase.

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

Stability of gas-phase tartaric acid anions investigated by quantum chemistry, mass spectrometry, and infrared spectroscopy.

In an effort to understand the chemical factors that stabilize dianions, experimental and theoretical studies on the stability of the tartrate dianion were performed. Quantum chemical calculations at the coupled cluster level reveal only a metastable state with a possible decomposition pathway (O(2)C-CH(OH)-CH(OH)-CO(2))(2-) → (O(2)C-CH(OH)-CH(OH))(•-) + CO(2) + e(-) explaining the observed gas-phase instability of this dianion. Further theoretical data were collected for the bare dianion, this molecule complexed to water, sodium, and a proton, in both the meso and l forms as well as for the uncomplexed radical anion and neutral diradical. The calculations suggest that the l-tartrate dianion is more thermodynamically stable than the dianion of the meso stereoisomer and that either dianion can be further stabilized by association with a separate species that can help to balance the charge of the molecular complex. Mass spectrometry was then used to measure the energy needed to initiate collisionally induced dissociation of the racemic tartrate dianion and for the proton and sodium adducts of both the racemic and meso form of this molecule. Infrared action spectra of the dianion stereoisomers complexed with sodium were also acquired to determine the influence of the metal ion on the vibrations of the dianions and validate the computationally predicted structures. These experimental data support the theoretical conclusions and highlight the instability of the bare tartrate dianion. From the experimental work, it could also be concluded that the pathway leading to dissociation is under kinetic control because the sodium adduct of the racemic stereoisomer dissociated at lower collisional energy, although it was calculated to be more stable, and that decomposition proceeded via C-C bond dissociation as computationally predicted. Taken together, these data provide insight into the gas-phase stability of the tartrate dianion and highlight the role of adducts in stabilizing this species.