One assay for all: exploring small molecule phosphorylation using amylose-polyiodide complexes.

We present a generic method for screening small molecule kinases for their acceptor specificity. The release of the reaction byproduct adenosine diphosphate (ADP) triggers a concentration-dependent formation of amylose from sucrose, by using the combined enzymatic action of sucrose synthase and glycogen synthase. Kinase activities could be quantified photometrically after the formation of a dark-blue amylose-polyiodide complex. We demonstrate that this method can be used to profile both known and novel nucleotide- and sugar-kinases for their substrate specificity. Using a facile and widely available methodology, the amylose-polyiodide small-molecule kinase assay presented herein has the potential to perform substrate screenings of small molecule kinases in a high-throughput manner.

Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus.

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

Discovery and Biochemical Characterization of UDP-Glucose Dehydrogenase from Granulibacter bethesdensis.

UDP-glucose dehydrogenases (EC 1.1.1.22) are responsible for the conversion of UDP-glucose to UDP-glucuronic acid, a key precursor in the biosynthesis of glycoconjugates. Herein we report the discovery and characterization of a UDPglucose dehydrogenase (GbUGD) from Granulibacter bethesdensis, a bacterium originally isolated from the lymph nodes of patients with chronic granulomatous disease (CGD). The recombinant form of the protein was expressed in high yield and the purified enzyme showed highest activity at 37°C/pH 9.0 and was strongly inhibited by Zn(2+) ions, sodium dodecyl sulfate (SDS) and urea. UDP-xylose, an allosteric feedback inhibitor, reduced significantly the activity of the enzyme. High activities were observed using the co-substrates UDP-glucose and NAD+, whereas no activity could be detected using other nucleotide sugars or NADP(+) as potential alternative substrates. The high activity combined with the simple purification procedure used make GbUGD a valuable new alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems.

Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila.

Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu(2+) and Zn(2+), and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.