Effects of Active-Site Modification and Quaternary Structure on the Regioselectivity of Catechol-O-Methyltransferase

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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Effect of E20D Substitution in the Active Site ofEscherichia coliInorganic Pyrophosphatase on Its Quaternary Structure and Catalytic Properties†

Biochemistry ◽

10.1021/bi952636m ◽

1996 ◽

Vol 35 (15) ◽

pp. 4662-4669 ◽

Cited By ~ 11

Author(s):

Sergei E. Volk ◽

Valerii Yu. Dudarenkov ◽

Jarmo Käpylä ◽

Vladimir N. Kasho ◽

Olga A. Voloshina ◽

...

Keyword(s):

Active Site ◽

Quaternary Structure ◽

Catalytic Properties

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Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase

Journal of Biological Chemistry ◽

10.1074/jbc.m111.234682 ◽

2011 ◽

Vol 286 (27) ◽

pp. 23877-23887 ◽

Cited By ~ 39

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.

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Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽

10.1182/blood.v67.4.1007.1007 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1007-1013 ◽

Cited By ~ 98

Author(s):

WJ Wallis ◽

DD Hickstein ◽

BR Schwartz ◽

CH June ◽

HD Ochs ◽

...

Keyword(s):

Monoclonal Antibody ◽

Active Site ◽

Bacterial Infections ◽

Quaternary Structure ◽

Alpha Subunit ◽

Human Neutrophils ◽

Glycoprotein Complex ◽

Dose Dependent Fashion ◽

Alpha Beta ◽

Dose Dependent

Abstract We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

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Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807954115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11623-E11632 ◽

Cited By ~ 11

Author(s):

Brandán Pedre ◽

David Young ◽

Daniel Charlier ◽

Álvaro Mourenza ◽

Leonardo Astolfi Rosado ◽

...

Keyword(s):

Active Site ◽

Promoter Region ◽

Structural Transition ◽

Quaternary Structure ◽

Antioxidant Response ◽

Active Site Residues ◽

Disulfide Formation ◽

Dimerization Interface ◽

H2o2 Sensing ◽

Key Residues

Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2. One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2. In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.

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Identification of a Subversive Substrate of Trichomonas vaginalis Purine Nucleoside Phosphorylase and the Crystal Structure of the Enzyme-Substrate Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m501843200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22318-22325 ◽

Cited By ~ 10

Author(s):

Yang Zang ◽

Wen-Hu Wang ◽

Shaw-Wen Wu ◽

Steven E. Ealick ◽

Ching C. Wang

Keyword(s):

Active Site ◽

Trichomonas Vaginalis ◽

Purine Nucleoside Phosphorylase ◽

Quaternary Structure ◽

Transmitted Disease ◽

Purine Nucleoside ◽

Purine Base ◽

Nucleoside Phosphorylase ◽

E Coli

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2′-deoxyadenosine (F-dAdo) is a “subversive substrate.” Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC50 values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2′-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 Å. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 Å as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.

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Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

Biochemical Journal ◽

10.1042/bj3400711 ◽

1999 ◽

Vol 340 (3) ◽

pp. 711-714 ◽

Cited By ~ 8

Author(s):

Sebastien FARNAUD ◽

Renée TATA ◽

Maninder K. SOHI ◽

Tommy WAN ◽

Paul R. BROWN ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Diffraction Analysis ◽

Active Site ◽

Quaternary Structure ◽

Cross Reactivity ◽

Polyclonal Antiserum ◽

Wild Type ◽

X Ray Diffraction ◽

Wild Type Enzyme ◽

X Ray

Wild-type and site-specific mutants C166S and C166A (Cys-166 → Ser and Cys-166 → Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5ʹ-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75 °) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

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Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014876 ◽

2020 ◽

Vol 295 (49) ◽

pp. 16863-16876

Author(s):

Rishi Raj ◽

Savitha Nadig ◽

Twinkal Patel ◽

Balasubramanian Gopal

Keyword(s):

Conformational Changes ◽

Active Site ◽

Staphylococcus Epidermidis ◽

Metal Ion ◽

Quaternary Structure ◽

Structural Similarity ◽

Endonuclease Activity ◽

Metal Cofactor ◽

Rna Maturation ◽

Metal Cofactors

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

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The structure of a nativel-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

Molecular BioSystems ◽

10.1039/c0mb00101e ◽

2011 ◽

Vol 7 (2) ◽

pp. 379-384 ◽

Cited By ~ 19

Author(s):

Dessislava Georgieva ◽

Mario Murakami ◽

Markus Perband ◽

Raghuvir Arni ◽

Christian Betzel

Keyword(s):

Amino Acid ◽

Active Site ◽

Quaternary Structure ◽

Acid Oxidase ◽

Divalent Ions ◽

Amino Acid Oxidase ◽

Structure Stabilization ◽

Vipera Ammodytes

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Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316019525 ◽

2017 ◽

Vol 73 (1) ◽

pp. 9-21 ◽

Cited By ~ 15

Author(s):

N. Mills-Davies ◽

D. Butler ◽

E. Norton ◽

D. Thompson ◽

M. Sarwar ◽

...

Keyword(s):

Active Site ◽

Quaternary Structure ◽

Early Stage ◽

Zinc Ion ◽

Salt Bridges ◽

Crystal Forms ◽

Hydrogen Bond Interactions ◽

Human Enzyme ◽

Tim Barrel ◽

High Resolution Structure

A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breiniget al.(2003),Nature Struct. Biol.10, 757–763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β)8barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termedAandB. In the native enzyme there are a number of distinct structural differences between theAandBmonomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, theAmonomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in theBsubunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophilePyrobaculum calidifontisis also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of theEscherichia coliALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzymeviahydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

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