Functional Annotation and Three-Dimensional Structure of an Incorrectly Annotated Dihydroorotase from cog3964 in the Amidohydrolase Superfamily

The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from <i>Agrobacterium tumefaciens</i> C58 and Oant2987 from <i>Ochrobactrum anthropi</i> ATCC 49188 were found to catalyze the hydrolysis of acetyl-(<i>R</i>)-mandelate and similar esters with values of <i>k</i>_cat/<i>K</i>_m that exceed 10⁵ M^–1 s^–1. These enzymes do not catalyze the deamination of adenine or the hydrolysis of dihydroorotate. Atu3266 was crystallized and the structure determined to a resolution of 2.62 Å. The protein folds as a distorted (β/α)₈ barrel and binds two zincs in the active site. The substrate profile was determined via a combination of computational docking to the three-dimensional structure of Atu3266 and screening of a highly focused library of potential substrates. The initial weak hit was the hydrolysis of <i>N</i>-acetyl-d-serine (<i>k</i>_cat/<i>K</i>_m = 4 M^–1 s^–1). This was followed by the progressive identification of acetyl-(<i>R</i>)-glycerate (<i>k</i>_cat/<i>K</i>_m = 4 × 10² M^–1 s^–1), acetyl glycolate (<i>k</i>_cat/<i>K</i>_m = 1.3 × 10⁴ M^–1 s^–1), and ultimately acetyl-(<i>R</i>)-mandelate (<i>k</i>_cat/<i>K</i>_m = 2.8 × 10⁵ M^–1 s^–1)

Additional file 1: Figure S1. of Induction of autophagy by ARHI (DIRAS3) alters fundamental metabolic pathways in ovarian cancer models

Growth of parental and ARHI-transfected SKOv3 and Hey cells. Effect of Dox treatment on the growth of parental and ARHI-transfected SKOv3 and Hey cells at 24 and 48 h. Western analysis of the effect of ARHI expression on LC3I and LC3II is also presented. Figure S2. Western analysis of GLUT1 expression following ARHI induction. Figure S3. Analysis of ARHI expression and autophagy markers during Atg5 knockdown. Effect of Atg5 knockdown on LC3I and LC3II levels during ARHI expression in SKOv3-ARHI cells. Immunofluorescence of SKOv3-ARHI cells transfected with GFP-LC3 following ARHI induction with and without Atg5 knockdown. Figure S4. Western analysis of LDH and CK expression following ARHI induction. Figure S5. Induction of ARHI expression in vivo. Expression of ARHI and LC3 in subcutaneous SKOv3-ARHI tumors at 24-72 h post-treatment with Dox. Figure S6. Expression of ACC and Phsopho-ACC by RPPA. Figure S7. Fractional 13C label incorporation from 5-13C-Gln in SKOv3-ARHI. The fractional incorporation of the glutamine 13C label into NMR-observable intracellular metabolites following induction of ARHI. (PDF 867 kb

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers

Radiolabeling of substrates with 2-[¹⁸F]­fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide–alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the ¹⁸F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[¹⁸F]­fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time