scholarly journals Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase

2011 ◽  
Vol 286 (27) ◽  
pp. 23877-23887 ◽  
Author(s):  
Sigrid Egger ◽  
Apirat Chaikuad ◽  
Kathryn L. Kavanagh ◽  
Udo Oppermann ◽  
Bernd Nidetzky
Keyword(s):  
Active Site ◽  
Quaternary Structure ◽  
Binding Domain ◽  
Side Chain ◽  
Reaction Conditions ◽  
Epithelial Tumor ◽  
Coenzyme Binding ◽  
Ternary Complex Formation ◽  
The Matrix

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.

The Study on the Preparation and Characterization of Crosslinked Polyvinyl Alcohol Microspheres

2013 ◽  
Vol 716 ◽  
pp. 337-342
Author(s):  
Yun Feng Yang ◽  
Bao Jiao Gao ◽  
Su Yu Liu
Keyword(s):  
Particle Size ◽  
Polyvinyl Alcohol ◽  
Crosslinking Agent ◽  
Influence Factors ◽  
Microemulsion Polymerization ◽  
Reaction Conditions ◽  
The Matrix ◽  
Inverse Microemulsion ◽  
Inverse Microemulsion Polymerization

Linear polyvinyl alcohol ( PVA ) as the matrix, Glutaraldehyde as crosslinking agent, the crosslinked polyvinyl alcohol microspheres ( CPVA ) was prepared by inverse microemulsion polymerization. Influence factors for forms and particle size of crosslinking spheres such as the ratio of oil and water, reaction temperature and time, crosslinking agent and catalyst use level, stirring speed. When reaction conditions changed, the particle size would change accordingly.

scholarly journals Characterization of SETD3 methyltransferase–mediated protein methionine methylation

2020 ◽  
Vol 295 (32) ◽  
pp. 10901-10910
Author(s):  
Shaobo Dai ◽  
Matthew V. Holt ◽  
John R. Horton ◽  
Clayton B. Woodcock ◽  
Anamika Patel ◽  
...  
Keyword(s):  
Active Site ◽  
Side Chain ◽  
Protein Methylation ◽  
Aromatic Rings ◽  
Set Domain ◽  
Histone Lysine ◽  
Histone Lysine Methyltransferase ◽  
Close Proximity ◽  
Lysine Methyltransferase

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310. Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.

scholarly journals Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4'- epimerase of Aeromonas hydrophila

BMB Reports ◽  
2010 ◽  
Vol 43 (6) ◽  
pp. 419-426 ◽  
Author(s):  
Shivani Agarwal ◽  
Neeraj Mishra ◽  
Shivangi Agarwal ◽  
Aparna Dixit
Keyword(s):  
Active Site ◽  
Aeromonas Hydrophila ◽  
Binding Pocket ◽  
Coenzyme Binding

scholarly journals Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

2015 ◽  
Vol 197 (8) ◽  
pp. 1360-1367 ◽  
Author(s):  
Amanda Ruprecht ◽  
Jaymie Maddox ◽  
Alexander J. Stirling ◽  
Nicole Visaggio ◽  
Stephen Y. K. Seah
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Side Chain ◽  
Side Chains ◽  
Content Type ◽  
Carbon Side Chain ◽  
Acyl Coa Dehydrogenases ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACTThe acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters ofMycobacterium tuberculosisandRhodococcus jostiiRHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of thecasCgene inR. jostiiRHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.IMPORTANCEWe report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, fromM. tuberculosisandRhodococcus jostiiRHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogenM. tuberculosiswithin host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

TEM characterization of reaction zone in a SiC fiber-reinforced titanium alloy

1988 ◽  
Vol 46 ◽  
pp. 738-739
Author(s):  
G. Das ◽  
R. E. Omlor
Keyword(s):  
Titanium Alloys ◽  
Reaction Zone ◽  
Carbon Layer ◽  
Fiber Reinforced ◽  
Reaction Products ◽  
Sic Fibers ◽  
Sic Fiber ◽  
The Matrix ◽  
Immense Potential

Fiber reinforced titanium alloys hold immense potential for applications in the aerospace industry. However, chemical reaction between the fibers and the titanium alloys at fabrication temperatures leads to the formation of brittle reaction products which limits their development. In the present study, coated SiC fibers have been used to evaluate the effects of surface coating on the reaction zone in the SiC/IMI829 system.IMI829 (Ti-5.5A1-3.5Sn-3.0Zr-0.3Mo-1Nb-0.3Si), a near alpha alloy, in the form of PREP powder (-35 mesh), was used a茸 the matrix. CVD grown AVCO SCS-6 SiC fibers were used as discontinuous reinforcements. These fibers of 142μm diameter contained an overlayer with high Si/C ratio on top of an amorphous carbon layer, the thickness of the coating being ∽ 1μm. SCS-6 fibers, broken into ∽ 2mm lengths, were mixed with IMI829 powder (representing < 0.1vol%) and the mixture was consolidated by HIP'ing at 871°C/0. 28GPa/4h.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

Sign in / Sign up

Export Citation Format

Share Document