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Dive into the research topics where Ellen W. Moomaw is active.

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Featured researches published by Ellen W. Moomaw.


Cell | 1996

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

Robert A Love; Hans E Parge; John A. Wickersham; Zdenek Hostomsky; Noriyuki Habuka; Ellen W. Moomaw; Tsuyoshi Adachi; Zuzana Hostomska

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.


PLOS ONE | 2013

Protein Similarity Networks Reveal Relationships among Sequence, Structure, and Function within the Cupin Superfamily

Richard Uberto; Ellen W. Moomaw

The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.


Biochemistry | 2009

Metal Dependence of Oxalate Decarboxylase Activity

Ellen W. Moomaw; Alexander Angerhofer; Patricia Moussatche; Andrew Ozarowski; Inés García-Rubio; Nigel G. J. Richards

Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO(2). These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.


Bioorganic & Medicinal Chemistry Letters | 1995

DESIGN, SYNTHESIS AND X-RAY CRYSTALLOGRAPHIC STUDIES OF NOVEL FKBP-12 LIGANDS

Robert E. Babine; T.M. Bleckman; C. R. Kissinger; Richard E. Showalter; Laura A. Pelletier; Cristina Lewis; Kathleen Tucker; Ellen W. Moomaw; Hans E Parge; J.Ernest Villafranca

Abstract Using the crystal structure of FKBP-12 a novel class of ligands were designed, prepared and evaluated. The crystal structure of the complex between 5 and FKBP-12 is reported.


Archives of Biochemistry and Biophysics | 2011

Characterization of Ceriporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris

Patricia Moussatche; Alexander Angerhofer; Witcha Imaram; Eric Hoffer; Kelsey Uberto; Christopher Brooks; Crystal Bruce; Daniel Sledge; Nigel G. J. Richards; Ellen W. Moomaw

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.


PLOS ONE | 2013

Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ellen W. Moomaw; Eric Hoffer; Patricia Moussatche; John C. Salerno; Morgan Grant; Bridget Immelman; Richard Uberto; Andrew Ozarowski; Alexander Angerhofer

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.


Molecular Plant Pathology | 2015

Fungal oxalate decarboxylase activity contributes to Sclerotinia sclerotiorum early infection by affecting both compound appressoria development and function

Xiaofei Liang; Ellen W. Moomaw; Jeffrey A. Rollins

Sclerotinia sclerotiorum pathogenesis requires the accumulation of high levels of oxalic acid (OA). To better understand the factors affecting OA accumulation, two putative oxalate decarboxylase (OxDC) genes (Ss-odc1 and Ss-odc2) were characterized. Ss-odc1 transcripts exhibited significant accumulation in vegetative hyphae, apothecia, early stages of compound appressorium development and during plant colonization. Ss-odc2 transcripts, in contrast, accumulated significantly only during mid to late stages of compound appressorium development. Neither gene was induced by low pH or exogenous OA in vegetative hyphae. A loss-of-function mutant for Ss-odc1 (Δss-odc1) showed wild-type growth, morphogenesis and virulence, and was not characterized further. Δss-odc2 mutants hyperaccumulated OA in vitro, were less efficient at compound appressorium differentiation and exhibited a virulence defect which could be fully bypassed by wounding the host plant prior to inoculation. All Δss-odc2 phenotypes were restored to the wild-type by ectopic complementation. An S. sclerotiorum strain overexpressing Ss-odc2 exhibited strong OxDC, but no oxalate oxidase activity. Increasing inoculum nutrient levels increased compound appressorium development, but not penetration efficiency, of Δss-odc2 mutants. Together, these results demonstrate differing roles for S. sclerotiorum OxDCs, with Odc2 playing a significant role in host infection related to compound appressorium formation and function.


PLOS ONE | 2017

Hydrogen peroxide inhibition of bicupin oxalate oxidase

John M. Goodwin; Hassan Rana; Joan Ndungu; Gaurab Chakrabarti; Ellen W. Moomaw

Oxalate oxidase is a manganese containing enzyme that catalyzes the oxidation of oxalate to carbon dioxide in a reaction that is coupled with the reduction of oxygen to hydrogen peroxide. Oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx) is the first fungal and bicupin enzyme identified that catalyzes this reaction. Potential applications of oxalate oxidase for use in pancreatic cancer treatment, to prevent scaling in paper pulping, and in biofuel cells have highlighted the need to understand the extent of the hydrogen peroxide inhibition of the CsOxOx catalyzed oxidation of oxalate. We apply a membrane inlet mass spectrometry (MIMS) assay to directly measure initial rates of carbon dioxide formation and oxygen consumption in the presence and absence of hydrogen peroxide. This work demonstrates that hydrogen peroxide is both a reversible noncompetitive inhibitor of the CsOxOx catalyzed oxidation of oxalate and an irreversible inactivator. The build-up of the turnover-generated hydrogen peroxide product leads to the inactivation of the enzyme. The introduction of catalase to reaction mixtures protects the enzyme from inactivation allowing reactions to proceed to completion. Circular dichroism spectra indicate that no changes in global protein structure take place in the presence of hydrogen peroxide. Additionally, we show that the CsOxOx catalyzed reaction with the three carbon substrate mesoxalate consumes oxygen which is in contrast to previous proposals that it catalyzed a non-oxidative decarboxylation with this substrate.


Biochemistry and biophysics reports | 2016

Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

Hassan Rana; Patricia Moussatche; Lis Souza Rocha; Sofiene Abdellaoui; Shelley D. Minteer; Ellen W. Moomaw

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔHapp). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.


Protein Science | 2017

PACMANS: A bioinformatically informed algorithm to predict, design, and disrupt protease-on-protease hydrolysis

Meghan C. Ferrall-Fairbanks; Zachary T. Barry; Maurizio Affer; Marc A. Shuler; Ellen W. Moomaw; Manu O. Platt

Multiple proteases in a system hydrolyze target substrates, but recent evidence indicates that some proteases will degrade other proteases as well. Cathepsin S hydrolysis of cathepsin K is one such example. These interactions may be uni‐ or bi‐directional and change the expected kinetics. To explore potential protease‐on‐protease interactions in silico, a program was developed for users to input two proteases: (1) the protease‐ase that hydrolyzes (2) the substrate, protease. This program identifies putative sites on the substrate protease highly susceptible to cleavage by the protease‐ase, using a sliding‐window approach that scores amino acid sequences by their preference in the protease‐ase active site, culled from MEROPS database. We call this PACMANS, Protease‐Ase Cleavage from MEROPS ANalyzed Specificities, and test and validate this algorithm with cathepsins S and K. PACMANS cumulative likelihood scoring identified L253 and V171 as sites on cathepsin K subject to cathepsin S hydrolysis. Mutations made at these locations were tested to block hydrolysis and validate PACMANS predictions. L253A and L253V cathepsin K mutants significantly reduced cathepsin S hydrolysis, validating PACMANS unbiased identification of these sites. Interfamilial protease interactions between cathepsin S and MMP‐2 or MMP‐9 were tested after predictions by PACMANS, confirming its utility for these systems as well. PACMANS is unique compared to other putative site cleavage programs by allowing users to define the proteases of interest and target, and can also be employed for non‐protease substrate proteins, as well as short peptide sequences.

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Richard Uberto

Kennesaw State University

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