Publications by authors named "Douglas C Goodwin"

24 Publications

  • Page 1 of 1

MRP.py: A Parametrizer of Post-Translationally Modified Residues.

J Chem Inf Model 2020 10 18;60(10):4424-4428. Epub 2020 Aug 18.

Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama36849-5312, United States.

MRP.py is a Python-based parametrization program for covalently modified amino acid residues for molecular dynamics simulations. Charge derivation is performed via an RESP charge fit, and force constants are obtained through rewriting of either protein or GAFF database parameters. This allows for the description of interfacial interactions between the modifed residue and protein. MRP.py is capable of working with a variety of protein databases. MRP.py's highly general and systematic method of obtaining parameters allows the user to circumvent the process of parametrizing the modified residue-protein interface. Two examples, a covalently bound inhibitor and covalent adduct consisting of modified residues, are provided in the Supporting Information.
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http://dx.doi.org/10.1021/acs.jcim.0c00472DOI Listing
October 2020

Slow-Binding Inhibition of Mycobacterium tuberculosis Shikimate Kinase by Manzamine Alkaloids.

Biochemistry 2018 08 31;57(32):4923-4933. Epub 2018 Jul 31.

Department of Drug Discovery and Development, Harrison School of Pharmacy , Auburn University , 4306 Walker Building , Auburn , Alabama 36849 , United States.

Tuberculosis represents a significant public health crisis. There is an urgent need for novel molecular scaffolds against this pathogen. We screened a small library of marine-derived compounds against shikimate kinase from Mycobacterium tuberculosis ( MtSK), a promising target for antitubercular drug development. Six manzamines previously shown to be active against M. tuberculosis were characterized as MtSK inhibitors: manzamine A (1), 8-hydroxymanzamine A (2), manzamine E (3), manzamine F (4), 6-deoxymanzamine X (5), and 6-cyclohexamidomanzamine A (6). All six showed mixed noncompetitive inhibition of MtSK. The lowest K values were obtained for 6 across all MtSK-substrate complexes. Time-dependent analyses revealed two-step, slow-binding inhibition. The behavior of 1 was typical; initial formation of an enzyme-inhibitor complex (EI) obeyed an apparent K of ∼30 μM with forward ( k) and reverse ( k) rate constants for isomerization to an EI* complex of 0.18 and 0.08 min, respectively. In contrast, 6 showed a lower K for the initial encounter complex (∼1.5 μM), substantially faster isomerization to EI* ( k = 0.91 min), and slower back conversion of EI* to EI ( k = 0.04 min). Thus, the overall inhibition constants, K*, for 1 and 6 were 10 and 0.06 μM, respectively. These findings were consistent with docking predictions of a favorable binding mode and a second, less tightly bound pose for 6 at MtSK. Our results suggest that manzamines, in particular 6, constitute a new scaffold from which drug candidates with novel mechanisms of action could be designed for the treatment of tuberculosis by targeting MtSK.
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http://dx.doi.org/10.1021/acs.biochem.8b00231DOI Listing
August 2018

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Biochim Biophys Acta Proteins Proteom 2018 May - Jun;1866(5-6):731-739. Epub 2018 Apr 12.

Department of Drug Discovery and Development, Harrison School of Pharmacy, 3306 Walker Building, Auburn University, Auburn, AL 36849, USA. Electronic address:

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M s. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.
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http://dx.doi.org/10.1016/j.bbapap.2018.04.007DOI Listing
July 2018

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase.

Bioorg Med Chem Lett 2018 02 5;28(4):802-808. Epub 2017 Dec 5.

Department of Drug Discovery and Development, Harrison School of Pharmacy, 4306 Walker Building, Auburn University, Auburn, AL 36849, USA. Electronic address:

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.
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http://dx.doi.org/10.1016/j.bmcl.2017.12.002DOI Listing
February 2018

Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

J Biol Chem 2017 11 27;292(45):18408-18421. Epub 2017 Sep 27.

From the Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849-5312,

KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against HO-induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert HO to O and HO only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of HO disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with HO Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates.
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http://dx.doi.org/10.1074/jbc.M117.791202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682954PMC
November 2017

Plasmodium falciparum Thioredoxin Reductase (PfTrxR) and Its Role as a Target for New Antimalarial Discovery.

Molecules 2015 Jun 22;20(6):11459-73. Epub 2015 Jun 22.

Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA.

The growing resistance to current antimalarial drugs is a major concern for global public health. The pressing need for new antimalarials has led to an increase in research focused on the Plasmodium parasites that cause human malaria. Thioredoxin reductase (TrxR), an enzyme needed to maintain redox equilibrium in Plasmodium species, is a promising target for new antimalarials. This review paper provides an overview of the structure and function of TrxR, discusses similarities and differences between the thioredoxin reductases (TrxRs) of different Plasmodium species and the human forms of the enzyme, gives an overview of modeling Plasmodium infections in animals, and suggests the role of Trx functions in antimalarial drug resistance. TrxR of Plasmodium falciparum is a central focus of this paper since it is the only Plasmodium TrxR that has been crystallized and P. falciparum is the species that causes most malaria cases. It is anticipated that the information summarized here will give insight and stimulate new directions in which research might be most beneficial.
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http://dx.doi.org/10.3390/molecules200611459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272602PMC
June 2015

Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials.

Perspect Medicin Chem 2015 15;7:9-20. Epub 2015 Mar 15.

Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, USA.

Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents.
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http://dx.doi.org/10.4137/PMC.S13212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362912PMC
April 2015

Inactivation of myeloperoxidase by benzoic acid hydrazide.

Arch Biochem Biophys 2015 Mar 14;570:14-22. Epub 2015 Feb 14.

Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, United States. Electronic address:

Myeloperoxidase (MPO) is expressed by myeloid cells for the purpose of catalyzing the formation of hypochlorous acid, from chloride ions and reaction with a hydrogen peroxide-charged heme covalently bound to the enzyme. Most peroxidase enzymes both plant and mammalian are inhibited by benzoic acid hydrazide (BAH)-containing compounds, but the mechanism underlying MPO inhibition by BAH compounds is largely unknown. Recently, we reported MPO inhibition by BAH and 4-(trifluoromethyl)-BAH was due to hydrolysis of the ester bond between MPO heavy chain glutamate 242 ((HC)Glu(242)) residue and the heme pyrrole A ring, freeing the heme linked light chain MPO subunit from the larger remaining heavy chain portion. Here we probed the structure and function relationship behind this ester bond cleavage using a panel of BAH analogs to gain insight into the constraints imposed by the MPO active site and channel leading to the buried protoporphyrin IX ring. In addition, we show evidence that destruction of the heme ring does not occur by tracking the heme prosthetic group and provide evidence that the mechanism of hydrolysis follows a potential attack of the (HC)Glu(242) carbonyl leading to a rearrangement causing the release of the vinyl-sulfonium linkage between (HC)Met(243) and the pyrrole A ring.
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http://dx.doi.org/10.1016/j.abb.2015.01.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779370PMC
March 2015

A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

Biochemistry 2015 Mar 23;54(8):1648-62. Epub 2015 Feb 23.

Department of Chemistry and Biochemistry, Auburn University , Auburn, Alabama 36849-5312, United States.

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.
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http://dx.doi.org/10.1021/bi501221aDOI Listing
March 2015

Development of an ESI-LC-MS-based assay for kinetic evaluation of Mycobacterium tuberculosis shikimate kinase activity and inhibition.

Anal Chem 2015 Feb 28;87(4):2129-36. Epub 2015 Jan 28.

Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University , 4306 Walker Building, Auburn, Alabama 36849, United States.

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.
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http://dx.doi.org/10.1021/ac503210nDOI Listing
February 2015

Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

Arch Biochem Biophys 2014 Feb 23;544:27-39. Epub 2013 Nov 23.

Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, USA. Electronic address:

Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation.
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http://dx.doi.org/10.1016/j.abb.2013.11.007DOI Listing
February 2014

Integral role of the I'-helix in the function of the "inactive" C-terminal domain of catalase-peroxidase (KatG).

Biochim Biophys Acta 2013 Jan 14;1834(1):362-71. Epub 2012 Aug 14.

Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, USA.

Catalase-peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I'-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I'-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG(C)). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG(N)). The analogous variants of KatG(C) showed a much more profound loss of function as evaluated by their ability to return KatG(N) to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG(N) reactivation, but with two substitutions, KatG(C) completely lost its capacity for the reactivation of KatG(N). These results suggest that the I'-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.
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http://dx.doi.org/10.1016/j.bbapap.2012.08.003DOI Listing
January 2013

Enhancing the peroxidatic activity of KatG by deletion mutagenesis.

J Inorg Biochem 2012 Nov 21;116:106-15. Epub 2012 Aug 21.

Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, United States.

Catalase-peroxidase (KatG) enzymes use a peroxidase active site to facilitate robust catalase activity, an ability all other members of its superfamily lack. KatG's have a Met-Tyr-Trp covalent adduct that is essential for catalatic but not peroxidatic turnover. The tyrosine (Y226 in E. coli KatG) is supplied by a large loop (LL1) that is absent from all other plant peroxidases. Elimination of Y226 from the KatG structure, either by site directed mutagenesis (i.e., Y226F KatG) or by deletion of larger portions of LL1 invariably eliminates catalase activity, but deletion variants were substantially more active as peroxidases, up to an order of magnitude. Moreover, the deletion variants were more resistant to H(2)O(2)-dependent inactivation than Y226F KatG. Stopped-flow evaluation of reactions of H(2)O(2) with Y226F KatG and the most peroxidase active deletion variant (KatG[Δ209-228]) produced highly similar rate constants for formation of compounds I and II, and about a four-fold faster formation of compound III for the deletion variant as opposed to Y226F. Conversely, single turnover experiments showed a 60-fold slower return of Y226F KatG to its ferric state in the presence of the exogenous electron donor 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) than was determined for KatG(Δ209-228). Our data suggest that the peroxidatic output of KatG cannot be optimized simply by elimination of catalase activity alone, but also requires modifications that increase electron transfer between exogenous electron donors and the heme prosthetic group.
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http://dx.doi.org/10.1016/j.jinorgbio.2012.08.002DOI Listing
November 2012

Stimulation of KatG catalase activity by peroxidatic electron donors.

Arch Biochem Biophys 2012 Sep 15;525(2):215-22. Epub 2012 Jun 15.

Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, USA.

Catalase-peroxidases (KatGs) use a peroxidase scaffold to support robust catalase activity, an ability no other member of its superfamily possesses. Because catalase turnover requires H(2)O(2) oxidation, whereas peroxidase turnover requires oxidation of an exogenous electron donor, it has been anticipated that the latter should inhibit catalase activity. To the contrary, we report peroxidatic electron donors stimulated catalase activity up to 14-fold, particularly under conditions favorable to peroxidase activity (i.e., acidic pH and low H(2)O(2) concentrations). We observed a "low-" and "high-K(M)" component for catalase activity at pH 5.0. Electron donors increased the apparent k(cat) for the "low-K(M)" component. During stimulated catalase activity, less than 0.008 equivalents of oxidized donor accumulated for every H(2)O(2) consumed. Several classical peroxidatic electron donors were effective stimulators of catalase activity, but pyrogallol and ascorbate showed little effect. Stopped-flow evaluation showed that a Fe(III)-O(2)(·-)-like intermediate dominated during donor-stimulated catalatic turnover, and this intermediate converted directly to the ferric state upon depletion of H(2)O(2). In this respect, the Fe(III)-O(2)(·-) -like species was more prominent and persistent than in the absence of the donor. These results point toward a much more central role for peroxidase substrates in the unusual catalase mechanism of KatG.
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http://dx.doi.org/10.1016/j.abb.2012.06.003DOI Listing
September 2012

Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase.

Biochem J 2011 Jan;433(1):163-74

Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour.
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http://dx.doi.org/10.1042/BJ20101353DOI Listing
January 2011

Substitution of strictly conserved Y111 in catalase-peroxidases: Impact of remote interdomain contacts on active site structure and catalytic performance.

J Inorg Biochem 2008 Sep 13;102(9):1819-24. Epub 2008 Jun 13.

Department of Chemistry and Biochemistry, Auburn University, AL 36849, United States.

Catalase-peroxidase function is strictly dependent on a gene-duplicated C-terminal domain. This domain no longer has a functioning active site, but from 25 to 30A away it is essential for preventing the coordination of an active site base (His106) to the heme. The mechanisms by which this distant structure supports active site function have not yet been elucidated. Tyr111 is a strictly conserved member of an interdomain H-bonding network that supports the loop connecting the N-terminal B (bearing His106) and C helices. Spectroscopic evaluation of the Tyr111Ala variant of KatG showed a substantial increase in hexa-coordinate low-spin heme, giving it the appearance of a transition between the wild type (primarily high-spin) and the N-terminal domain alone (pure low-spin). Concomitant with the spectral changes was decreased activity compared to the wild type enzyme, suggesting that Tyr111 does have a role in preventing His106 coordination. Substitution of Tyr111 diminishes catalase activity more substantially than peroxidase activity. Such an effect cannot be explained by His106 coordination alone, suggesting that these interdomain interactions may help tune the catalase-peroxidase active site for bifunctionality.
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http://dx.doi.org/10.1016/j.jinorgbio.2008.06.002DOI Listing
September 2008

The kinetic properties producing the perfunctory pH profiles of catalase-peroxidases.

Biochim Biophys Acta 2008 Jun 21;1784(6):900-7. Epub 2008 Mar 21.

Department of Chemistry and Biochemistry, Auburn University, AL 36849, USA.

Many structure-function relationship studies performed on the catalase-peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of catalase-peroxidase function, we undertook a more exhaustive evaluation of catalase-peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent pK(a)s for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.
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http://dx.doi.org/10.1016/j.bbapap.2008.03.008DOI Listing
June 2008

Interactions between nitric oxide and peroxynitrite during prostaglandin endoperoxide H synthase-1 catalysis: a free radical mechanism of inactivation.

Free Radic Biol Med 2007 Apr 9;42(7):1029-38. Epub 2007 Jan 9.

Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.

Peroxynitrite (ONOO(-)) can serve either as a peroxide substrate or as an inactivator of prostaglandin endoperoxide H synthase-1 (PGHS-1). Herein, the mechanism of PGHS-1 inactivation by ONOO(-) and the modulatory role that nitric oxide (*NO) plays in this process were studied. PGHS-1 reacted with ONOO(-) with a second-order rate constant of 1.7 x 10(7) M(-1) s(-1) at pH 7.0 and 8 degrees C. In the absence of substrates, the enzyme was dose-dependently inactivated by ONOO(-) in parallel with 3-nitrotyrosine formation. However, when PGHS-1 was incubated with ONOO(-) in the presence of substrates, the direct reaction with ONOO(-) was less relevant and ONOO(-)-derived radicals became involved in enzyme inactivation. Bicarbonate at physiologically relevant concentrations enhanced PGHS-1 inactivation and nitration by ONOO(-), further supporting a free radical mechanism. Importantly, *NO (0.4-1.5 microM min(-1)) was able to spare the peroxidase activity of PGHS-1 but it enhanced ONOO(-)-mediated inactivation of cyclooxygenase. The observed differential effects of *NO on ONOO(-)-mediated PGHS-1 inactivation emphasize a novel aspect of the complex modulatory role that *NO plays during inflammatory processes. We conclude that ONOO(-)-derived radicals inactivate both peroxidase and cyclooxygenase activities of PGHS-1 during enzyme turnover. Finally, our results reconcile the proposed alternative effects of ONOO(-) on PGHS-1 (activation versus inactivation).
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http://dx.doi.org/10.1016/j.freeradbiomed.2007.01.009DOI Listing
April 2007

Catalase-peroxidase active site restructuring by a distant and "inactive" domain.

Biochemistry 2006 Jun;45(23):7113-21

Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849-5312, USA.

Catalase-peroxidases are composed of two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. The C-terminal domain does not bind heme, has no catalytic activity, and is separated from the active site by >30 A. Nevertheless, without the C-terminal domain, the N-terminal domain exhibits neither catalase nor peroxidase activity due to the apparent coordination of the distal histidine to the heme iron. Here we report the ability of the separately expressed and isolated C-terminal domain (KatG(C)) to restructure the N-terminal domain (KatG(N)) to its bifunctional conformation. Addition of equimolar KatG(C) to KatG(N) decreased the hexacoordinate low-spin heme complex and increased the high-spin species (pentacoordinate and hexacoordinate). EPR spectra of the domain mixture showed a distribution between high-spin species nearly identical to that of wild-type KatG. The CD spectrum for the 1:1 physical mixture of the domains was identical to an arithmetic composite of individual spectra for KatG(N) and KatG(C). Both physical and arithmetic mixtures were nearly identical to the spectrum for wild-type KatG, suggesting that major shifts in secondary structure did not accompany active site reconfiguration. With the shift in heme environment, the parallel return of catalase and peroxidase activity was observed. Inclusion of bovine serum albumin instead of KatG(C) produced no activity, indicating that specific interdomain interactions were required to reestablish the bifunctional active site. Apparent constants for reactivation (k(react) approximately 4 x 10(-3) min(-1)) indicate that a slow process like movement of established structural elements may precede the restructuring of the heme environment and return of catalytic activity.
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http://dx.doi.org/10.1021/bi052392yDOI Listing
June 2006

Properties of catalase-peroxidase lacking its C-terminal domain.

Biochem Biophys Res Commun 2004 Jul;320(3):833-9

Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, USA.

Catalase-peroxidases have a two-domain structure. The N-terminal domain contains the bifunctional active site, but the function of the C-terminal domain is unknown. We produced catalase-peroxidase containing only its N-terminal domain (KatG(Nterm)). Removal of the C-terminal domain did not result in unexpected changes in secondary structure as evaluated by CD, but KatG(Nterm) had neither catalase nor peroxidase activity. Partial recovery of both activities was achieved by incubating KatG(Nterm) with the separately expressed and isolated KatG C-terminal domain. Spectroscopic measurements revealed a shift in heme environment from a mixture of high-spin species (wtKatG) to exclusively hexacoordinate, low-spin (KatG(Nterm)). Moreover, a > 1000-fold lower kon for CN- binding was observed for KatG(Nterm). EPR spectra for KatG(Nterm) and the results of site-specific substitution of active site histidines suggested that the distal histidine was the sixth ligand. Thus, one important role for the C-terminal domain may be to support the architecture of the active site, preventing heme ligation by this catalytically essential residue.
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http://dx.doi.org/10.1016/j.bbrc.2004.06.026DOI Listing
July 2004

Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality.

Biochem Biophys Res Commun 2004 Jun;318(4):970-6

Department of Chemistry and Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5312, USA.

The loop connecting the F and G helices of catalase-peroxidases contains a approximately 35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases. These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity. Thus, the FG insertion may serve a role in catalase-peroxidase bifunctionality, despite its peripheral location relative to the active site. We produced a variant of Escherichia coli catalase-peroxidase (KatG) lacking its FG insertion (KatG(DeltaFG)). Absorption spectra indicated the heme environment of KatG(DeltaFG) was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity. In contrast, the deletion reduced peroxidase activity by only 50%. Kinetic parameters for the peroxidase and residual catalase activities of KatG(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2. The structure also appears to regulate access of electron donors to the active site.
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http://dx.doi.org/10.1016/j.bbrc.2004.04.130DOI Listing
June 2004

System for the expression of recombinant hemoproteins in Escherichia coli.

Protein Expr Purif 2004 May;35(1):76-83

Department of Chemistry, Auburn University, Auburn, AL 36849-5312, USA.

Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E. coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, catalase-peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived catalase-peroxidase were the same as those observed for catalase-peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function.
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http://dx.doi.org/10.1016/j.pep.2003.12.001DOI Listing
May 2004

Properties of a novel periplasmic catalase-peroxidase from Escherichia coli O157:H7.

Arch Biochem Biophys 2004 Jan;421(1):166-74

Department of Chemistry, Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5312, USA.

A subset of catalase-peroxidases are distinguished by their periplasmic location and their expression by pathogens. Kinetic and spectral properties have not been reported for any of these enzymes. We report the cloning, expression, isolation, and characterization of KatP, a periplasmic catalase-peroxidase from Escherichia coli O157:H7. Absorption spectra indicated a mixture of heme states dominated by the pentacoordinate and hexacoordinate high-spin forms. Apparent k(cat) values for catalase (1.8x10(4) s(-1)) and peroxidase (77 s(-1)) activities were greater than those of other catalase-peroxidases. However, apparent K(M) values for H2O2 were also higher (27 mM for catalase and 3 mM for peroxidase). Ferric KatP reacted with peracetic acid to form compound I (8.8x10(3) M(-1) s(-1)) and with CN(-) to form a ferri-cyano complex (3.9x10(5) M(-1) s(-1)) consistent with other catalase-peroxidases. The isolation and characterization of KatP opens new avenues to explore mechanisms by which the periplasmic catalase-peroxidases may contribute to bacterial virulence.
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http://dx.doi.org/10.1016/j.abb.2003.10.020DOI Listing
January 2004

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies.

Arch Biochem Biophys 2003 Sep;417(1):18-26

Department of Chemistry, Auburn University, Auburn, AL 36849-5312, USA.

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.
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http://dx.doi.org/10.1016/s0003-9861(03)00321-7DOI Listing
September 2003
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