Maria Pandelia - Academia.edu (original) (raw)
Papers by Maria Pandelia
The FASEB Journal, 2015
Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the i... more Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n-octanoyl chain on a lipoyl carrier protein (LCP). ...
HBx is the smallest gene product of the Hepatitis B virus (HBV) and an oncogenic stimulus in chro... more HBx is the smallest gene product of the Hepatitis B virus (HBV) and an oncogenic stimulus in chronic infections leading to liver disease. HBx interacts and interferes with numerous cellular processes, but its modes of action remain poorly understood. It has been invoked that HBx employs nucleotide hydrolysis to regulate molecular pathways or protein−protein interactions. In the present study, we reinvestigate the (d)NTP hydrolysis of recombinant HBx to explore its potential as a biochemical probe for antiviral studies. For our investigations, we employed existing soluble constructs (i.e., GST-HBx, MBP-HBx) and engineered new fusion proteins (i.e., DsbC-HBx, NusA-HBx), which are shown to serve as better systems for in vitro research. We performed mutational scanning of the computationally predicted NTP-binding domain, which includes residues associated with clinical cases. Steady-state and end-point activity assays, in tandem with mass-spectrometric analyses, reveal that the observed hydrolysis of all alleged HBx substrates, ATP, dATP, and GTP, is contingent on the presence of the GroEL chaperone, which preferentially copurifies as a contaminant with GST-HBx and MBP-HBx. Collectively, our findings provide new technical standards for recombinant HBx studies and reveal that nucleotide hydrolysis is not an operant mechanism by which HBx contributes to viral HBV carcinogenesis.
![Research paper thumbnail of HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family](https://attachments.academia-assets.com/91842457/thumbnails/1.jpg)
](Cyclic dinucleotides are signaling molecules that modulate many processes, including immune respo... more Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metaldependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3′3′ c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized Fe II Fe III state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent k cat and K M (but not k cat /K M), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.
Biochemistry, 2022
Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by ... more Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by cleaving foreign DNA. In addition, they impact bacterial physiology by regulating pathogenicity and virulence, making them key players in adaptability and evolution. The signature nuclease Cas3 is a phosphodiesterase belonging to the HD-domain metalloprotein superfamily. By directing specific metal incorporation, we map a promiscuous metal ion cofactor profile for Cas3 from Thermobifida fusca (Tf). Tf Cas3 affords significant ssDNA cleavage with four homo-dimetal centers (Fe 2+ , Co 2+ , Mn 2+ , and Ni 2+), while the diferrous form is the most active and likely biologically relevant in vivo. Electron paramagnetic resonance (EPR) spectroscopy and Mössbauer spectroscopy show that the diiron cofactor can access three redox forms, while the diferrous form can be readily obtained with mild reductants. We further employ EPR and Mössbauer on Fe-enriched proteins to establish that Cas3″ enzymes harbor a dinuclear cofactor, which was not previously confirmed. We demonstrate that the ancillary His ligand is critical for efficient ssDNA cleavage but not for diiron assembly or small molecule hydrolysis. We further explore the ability of Cas3 to hydrolyze cyclic mononucleotides and show that Tf Cas3 hydrolyzes 2′3′-cAMP with catalytic efficiency comparable to that of the conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2′3′-cylic phosphodiester bonds at RNA 3′-termini.
Journal of Biological Chemistry, 2022
The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main et... more The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.
![Research paper thumbnail of ChlR of Synechococcus sp. PCC 7002 is a transcription activator that uses an oxygen-sensitive [4Fe-4S] cluster to control genes involved in pigment biosynthesis](https://attachments.academia-assets.com/85185772/thumbnails/1.jpg)
](Journal of Biological Chemistry, 2014
Biochimica et Biophysica Acta - Proteins and Proteomics, 2018
The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2)... more The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate dcamphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. Cytochrome P450 cam (CYP101A1) is a heme-containing monooxygenase that catalyzes the stereospecific 5-exo hydroxylation of the substrate camphor by molecular oxygen, O 2 (Scheme I). The overall reaction involves insertion of one oxygen atom into the 5-exo C-H bond of camphor 1 to form 5-exo-hydroxycamphor 2, with concomitant reduction of the other oxygen atom to water. The oxygen reduction requires two electrons, provided by the iron-sulfur protein putidaredoxin (Pdx) via two separate one-electron shuttles from the flavoenzyme putidaredoxin reductase (PdR). PdR catalyzes the two-electron oxidation of NADH, the ultimate source of reducing equivalents in the P450 reaction cycle.
Biochemistry
Materials. Yeast extract, tryptone, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased... more Materials. Yeast extract, tryptone, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from DOT Scientific. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycerol, and imidazole, were purchased from Fisher Scientific. Kanamycin was purchased from Teknova. Sodium chloride (NaCl), sodium hydroxide (NaOH), ammonium chloride (NH 4 Cl) and hydrochloric acid (HCl) were purchased from EMD Millipore. Dithiothreitol (DTT) and 1,4-dihydroxynapthalene (NQ) were purchased from Alfa Aeser. Ni II-nitrilotriacetic acid agarose (Ni-NTA) resin and 96-well crystallography screens were purchased from Qiagen. All other chemicals used were purchased from Sigma Aldrich. Oligonucleotide primers were purchased from Integrated DNA Technologies. PCR reagents, restriction enzymes/buffers, and T4 DNA ligase were purchased from New England Biolabs. Escherichia coli (Ec) DH5 α, BL21 (DE3), and the pET-28a(+) vector were purchased from Novagen. All crystallography supplies were purchased from Qiagen and Hampton Research.
eLife
The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni... more The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging ...
Biochemistry
Abstract We have synthesized several novel biomimetic interfacial structures through the covalent... more Abstract We have synthesized several novel biomimetic interfacial structures through the covalent growth of individual layers on oxide-based, electrochemically active substrates (eg gold oxide, ITO, boron-doped diamond). Our goal is to deposit selected biomolecules on ...
eLife
Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of th... more Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of the genes and enzymes responsible for this metabolism are unknown. Recently, we linked the two-gene ‘cardiac glycoside reductase’ (cgr) operon encoded by the gut Actinobacterium Eggerthella lenta to inactivation of the cardiac medication and plant natural product digoxin. Here, we compared the genomes of 25 E. lenta strains and close relatives, revealing an expanded 8-gene cgr-associated gene cluster present in all digoxin metabolizers and absent in non-metabolizers. Using heterologous expression and in vitro biochemical characterization, we discovered that a single flavin- and [4Fe-4S] cluster-dependent reductase, Cgr2, is sufficient for digoxin inactivation. Unexpectedly, Cgr2 displayed strict specificity for digoxin and other cardenolides. Quantification of cgr2 in gut microbiomes revealed that this gene is widespread and conserved in the human population. Together, these results demons...
Science (New York, N.Y.), Mar 23, 2018
Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for cor... more Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.
Biochemistry, May 2, 2018
A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae (Fj) differs fundamentally from k... more A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae (Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its β subunit shares with Ib counterparts the requirements for manganese(II) and superoxide for activation, but it does not require the superoxide-supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj β has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj β directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) βs. This tendency makes Fj β unusually robust, as the II/II form can readily be r...
Biochimica et biophysica acta, 2017
The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2)... more The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to...
Journal of the American Chemical Society, Aug 10, 2016
Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme invol... more Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with (13)C and (2)H isotopically labeled SAMCA support a π-complex between the C═C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S](+) cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our c...
Biochemistry, Jan 3, 2016
The prevalence of multiple- and extensively-drug-resistant strains of Mycobacterium tuberculosis ... more The prevalence of multiple- and extensively-drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein, and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus and Thermosynechococcus elongatus have been conducted, but the enz...
![Research paper thumbnail of Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster](https://attachments.academia-assets.com/71062944/thumbnails/1.jpg)
](Journal of the American Chemical Society, 2015
The FASEB Journal, 2015
Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the i... more Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n-octanoyl chain on a lipoyl carrier protein (LCP). ...
HBx is the smallest gene product of the Hepatitis B virus (HBV) and an oncogenic stimulus in chro... more HBx is the smallest gene product of the Hepatitis B virus (HBV) and an oncogenic stimulus in chronic infections leading to liver disease. HBx interacts and interferes with numerous cellular processes, but its modes of action remain poorly understood. It has been invoked that HBx employs nucleotide hydrolysis to regulate molecular pathways or protein−protein interactions. In the present study, we reinvestigate the (d)NTP hydrolysis of recombinant HBx to explore its potential as a biochemical probe for antiviral studies. For our investigations, we employed existing soluble constructs (i.e., GST-HBx, MBP-HBx) and engineered new fusion proteins (i.e., DsbC-HBx, NusA-HBx), which are shown to serve as better systems for in vitro research. We performed mutational scanning of the computationally predicted NTP-binding domain, which includes residues associated with clinical cases. Steady-state and end-point activity assays, in tandem with mass-spectrometric analyses, reveal that the observed hydrolysis of all alleged HBx substrates, ATP, dATP, and GTP, is contingent on the presence of the GroEL chaperone, which preferentially copurifies as a contaminant with GST-HBx and MBP-HBx. Collectively, our findings provide new technical standards for recombinant HBx studies and reveal that nucleotide hydrolysis is not an operant mechanism by which HBx contributes to viral HBV carcinogenesis.
Cyclic dinucleotides are signaling molecules that modulate many processes, including immune respo... more Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metaldependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3′3′ c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized Fe II Fe III state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent k cat and K M (but not k cat /K M), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.
Biochemistry, 2022
Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by ... more Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by cleaving foreign DNA. In addition, they impact bacterial physiology by regulating pathogenicity and virulence, making them key players in adaptability and evolution. The signature nuclease Cas3 is a phosphodiesterase belonging to the HD-domain metalloprotein superfamily. By directing specific metal incorporation, we map a promiscuous metal ion cofactor profile for Cas3 from Thermobifida fusca (Tf). Tf Cas3 affords significant ssDNA cleavage with four homo-dimetal centers (Fe 2+ , Co 2+ , Mn 2+ , and Ni 2+), while the diferrous form is the most active and likely biologically relevant in vivo. Electron paramagnetic resonance (EPR) spectroscopy and Mössbauer spectroscopy show that the diiron cofactor can access three redox forms, while the diferrous form can be readily obtained with mild reductants. We further employ EPR and Mössbauer on Fe-enriched proteins to establish that Cas3″ enzymes harbor a dinuclear cofactor, which was not previously confirmed. We demonstrate that the ancillary His ligand is critical for efficient ssDNA cleavage but not for diiron assembly or small molecule hydrolysis. We further explore the ability of Cas3 to hydrolyze cyclic mononucleotides and show that Tf Cas3 hydrolyzes 2′3′-cAMP with catalytic efficiency comparable to that of the conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2′3′-cylic phosphodiester bonds at RNA 3′-termini.
Journal of Biological Chemistry, 2022
The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main et... more The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.
Journal of Biological Chemistry, 2014
Biochimica et Biophysica Acta - Proteins and Proteomics, 2018
The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2)... more The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate dcamphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. Cytochrome P450 cam (CYP101A1) is a heme-containing monooxygenase that catalyzes the stereospecific 5-exo hydroxylation of the substrate camphor by molecular oxygen, O 2 (Scheme I). The overall reaction involves insertion of one oxygen atom into the 5-exo C-H bond of camphor 1 to form 5-exo-hydroxycamphor 2, with concomitant reduction of the other oxygen atom to water. The oxygen reduction requires two electrons, provided by the iron-sulfur protein putidaredoxin (Pdx) via two separate one-electron shuttles from the flavoenzyme putidaredoxin reductase (PdR). PdR catalyzes the two-electron oxidation of NADH, the ultimate source of reducing equivalents in the P450 reaction cycle.
Biochemistry
Materials. Yeast extract, tryptone, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased... more Materials. Yeast extract, tryptone, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from DOT Scientific. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycerol, and imidazole, were purchased from Fisher Scientific. Kanamycin was purchased from Teknova. Sodium chloride (NaCl), sodium hydroxide (NaOH), ammonium chloride (NH 4 Cl) and hydrochloric acid (HCl) were purchased from EMD Millipore. Dithiothreitol (DTT) and 1,4-dihydroxynapthalene (NQ) were purchased from Alfa Aeser. Ni II-nitrilotriacetic acid agarose (Ni-NTA) resin and 96-well crystallography screens were purchased from Qiagen. All other chemicals used were purchased from Sigma Aldrich. Oligonucleotide primers were purchased from Integrated DNA Technologies. PCR reagents, restriction enzymes/buffers, and T4 DNA ligase were purchased from New England Biolabs. Escherichia coli (Ec) DH5 α, BL21 (DE3), and the pET-28a(+) vector were purchased from Novagen. All crystallography supplies were purchased from Qiagen and Hampton Research.
eLife
The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni... more The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging ...
Biochemistry
Abstract We have synthesized several novel biomimetic interfacial structures through the covalent... more Abstract We have synthesized several novel biomimetic interfacial structures through the covalent growth of individual layers on oxide-based, electrochemically active substrates (eg gold oxide, ITO, boron-doped diamond). Our goal is to deposit selected biomolecules on ...
eLife
Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of th... more Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of the genes and enzymes responsible for this metabolism are unknown. Recently, we linked the two-gene ‘cardiac glycoside reductase’ (cgr) operon encoded by the gut Actinobacterium Eggerthella lenta to inactivation of the cardiac medication and plant natural product digoxin. Here, we compared the genomes of 25 E. lenta strains and close relatives, revealing an expanded 8-gene cgr-associated gene cluster present in all digoxin metabolizers and absent in non-metabolizers. Using heterologous expression and in vitro biochemical characterization, we discovered that a single flavin- and [4Fe-4S] cluster-dependent reductase, Cgr2, is sufficient for digoxin inactivation. Unexpectedly, Cgr2 displayed strict specificity for digoxin and other cardenolides. Quantification of cgr2 in gut microbiomes revealed that this gene is widespread and conserved in the human population. Together, these results demons...
Science (New York, N.Y.), Mar 23, 2018
Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for cor... more Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.
Biochemistry, May 2, 2018
A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae (Fj) differs fundamentally from k... more A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae (Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its β subunit shares with Ib counterparts the requirements for manganese(II) and superoxide for activation, but it does not require the superoxide-supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj β has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj β directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) βs. This tendency makes Fj β unusually robust, as the II/II form can readily be r...
Biochimica et biophysica acta, 2017
The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2)... more The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to...
Journal of the American Chemical Society, Aug 10, 2016
Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme invol... more Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with (13)C and (2)H isotopically labeled SAMCA support a π-complex between the C═C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S](+) cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our c...
Biochemistry, Jan 3, 2016
The prevalence of multiple- and extensively-drug-resistant strains of Mycobacterium tuberculosis ... more The prevalence of multiple- and extensively-drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein, and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus and Thermosynechococcus elongatus have been conducted, but the enz...
Journal of the American Chemical Society, 2015