Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase.

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

Thiamine pyrophosphate stimulates acetone activation by Desulfococcus biacutus as monitored by a fluorogenic ATP analogue.

Acetone can be degraded by aerobic and anaerobic microorganisms. Studies with the strictly anaerobic sulfate-reducing bacterium Desulfococcus biacutus indicate that acetone degradation by these bacteria starts with an ATP-dependent carbonylation reaction leading to acetoacetaldehyde as the first reaction product. The reaction represents the second example of a carbonylation reaction in the biochemistry of strictly anaerobic bacteria, but the exact mechanism and dependence on cofactors are still unclear. Here, we use a novel fluorogenic ATP analogue to investigate its mechanism. We find that thiamine pyrophosphate is a cofactor of this ATP-dependent reaction. The products of ATP cleavage are AMP and pyrophosphate, providing first insights into the reaction mechanism by indicating that the reaction proceeds without intermediate formation of acetone enol phosphate.

Synthesis and fluorescence characteristics of ATP-based FRET probes.

Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes.

Carbonylation as a key reaction in anaerobic acetone activation by Desulfococcus biacutus.

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO2. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg(-1) protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.