Katrin GRage | Massey University (original) (raw)
Papers by Katrin GRage
![Research paper thumbnail of Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two bound S-adenosyl-L-methionines](https://attachments.academia-assets.com/46440163/thumbnails/1.jpg)
](The Journal of biological chemistry, Jan 12, 2005
The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidas... more The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S] 1؉ cluster in two slightly different conformations. Mö ssbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S] 2؉ cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of ␦ ؍ 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a ␦ ؍ 0.57 (3) mm/s, which shifted to a ␦ ؍ 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mu-tants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.
…, 2009
Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for ... more Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.
Applied and Environmental Microbiology, 2009
Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty aci... more Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisincontrolled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granuleassociated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
Applied and Environmental Microbiology, 2011
A novel approach to produce purified recombinant proteins was established. The target protein is ... more A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.
Bioconjugate Chemistry, 2008
Recombinant production and, in particular, immobilization of antibody fragments onto carrier mate... more Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.
During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) cataly... more During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.
The Journal of biological chemistry, Jan 12, 2005
The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidas... more The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S] 1؉ cluster in two slightly different conformations. Mö ssbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S] 2؉ cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of ␦ ؍ 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a ␦ ؍ 0.57 (3) mm/s, which shifted to a ␦ ؍ 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mu-tants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.
…, 2009
Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for ... more Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.
Applied and Environmental Microbiology, 2009
Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty aci... more Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisincontrolled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granuleassociated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
Applied and Environmental Microbiology, 2011
A novel approach to produce purified recombinant proteins was established. The target protein is ... more A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.
Bioconjugate Chemistry, 2008
Recombinant production and, in particular, immobilization of antibody fragments onto carrier mate... more Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.
During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) cataly... more During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.