Papers by Catherine Drennan
Metallomics
Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for li... more Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complica...
eLife, 2020
The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze diffic... more The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.
Journal of the American Chemical Society, 2018
TYW1 is a radical S-adenosyl-L-methionine (SAM) enzyme that catalyzes the condensation of pyruvat... more TYW1 is a radical S-adenosyl-L-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine to form the posttranscriptional modification, 4-demethylwyosine, in situ on transfer RNA (tRNA). Two mechanisms have been proposed for this transformation, with one of the possible mechanisms invoking a Schiff base intermediate formed between a conserved lysine residue and pyruvate. Utilizing a combination of mass spectrometry and X-ray crystallography, we have obtained evidence to support the formation of a Schiff base lysine adduct in TYW1. When 13 C labeled pyruvate is used, the mass shift of the adduct matches that of the labeled pyruvate, indicating that pyruvate is the source of the adduct. Furthermore, a crystal structure of TYW1 provides visualization of the Schiff base lysine−pyruvate adduct, which is positioned directly adjacent to the auxiliary [4Fe−4S] cluster. The adduct coordinates the unique iron of the auxiliary cluster through the lysine nitrogen and a carboxylate oxygen, reminiscent of how the radical SAM [4Fe−4S] cluster is coordinated by SAM. The structure provides insight into the binding site for tRNA and further suggests how radical SAM chemistry can be combined with Schiff base chemistry for RNA modification.
Science (New York, N.Y.), Dec 8, 2017
Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in... more Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from (HEPD), and find that it displays the same motif. HEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus.
Angewandte Chemie (International ed. in English), Jan 15, 2018
The electron-rich isonitrile is an important functionality in bioactive natural products, but its... more The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, members of which are critical for the virulence of pathogenic mycobacteria.
The Journal of biological chemistry, Jan 25, 2016
Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme... more Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Å resolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ∼33 Å across an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within ...
Cancer & Metabolism, 2015
Background: The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region... more Background: The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein. Results: The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure. Conclusions: Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2014
Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent protein... more Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conve...
Structure, 1996
Determination of the structure of intact methylmalonyl-CoA mutase from Propionibacterium shermani... more Determination of the structure of intact methylmalonyl-CoA mutase from Propionibacterium shermanii, and comparisons with the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli, afford a first glimpse at the similarities and distinctions between the two principal classes of B 12-dependent enzymes: the mutases and the methyltransferases.
Proceedings of the National Academy of Sciences, 1996
Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms ... more Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in fr...
Proceedings of the National Academy of Sciences, 2007
The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron o... more The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product.
Journal of the American Chemical Society, 2009
CytC3, a member of the recently discovered class of nonheme Fe(II) and R-ketoglutarate (RKG)depen... more CytC3, a member of the recently discovered class of nonheme Fe(II) and R-ketoglutarate (RKG)dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, γ,γ-dichloroaminobutyrate. Unlike the majority of the Fe(II)-RKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without RKG/Fe(II) have been solved to 2.2 Å resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound FesCl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.
Journal of Biological Chemistry, 2007
MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B 12... more MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B 12-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central ␣/-core G domain that is typical of the GTPase family, as well as ␣-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP-and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease. * This work was supported in part by National Institutes of Health Grants GM65337 (to C. L. D.) and DK45776 (to R. B.), the Ralph L. Evans (1948) Endowment Fund (to S. A. M.), the Cathy M. Comeau Memorial Fund (to S. A. M.), and the MIT Center for Environmental Health Sciences NIEH P30 ES002109. Synchrotron facilities are supported by the United States Department of Energy and the National Institutes of Health. Use of IMCA-CAT beam line 17-ID was supported by the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation at the University of Chicago. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2qm7 and 2qm8) have been deposited in the Protein Data Bank,
Journal of Biological Chemistry, 2011
Journal of Bacteriology, 2005
Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaero... more Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF hom...
Biophysical Journal, 2009
Biochemistry, 2011
To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold ... more To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.
Proceedings of the National Academy of Sciences, 2011
Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotid... more Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit ( α 2 ) and the radical-generation subunit ( β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli . Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β ...
Structural Studies of Allosteric Regulation in the Class Ia Ribonucleotide Reductase from Escheri... more Structural Studies of Allosteric Regulation in the Class Ia Ribonucleotide Reductase from Escherichia coli by
ACS Chemical Biology, 2019
Leishmaniases affect the poorest people on earth and have no effective drug therapy. Here, we pre... more Leishmaniases affect the poorest people on earth and have no effective drug therapy. Here, we present the crystal structure of the mitochondrial isoform of class I fumarate hydratase (FH) from Leishmania major and compare it to the previously determined cytosolic Leishmania major isoform. We further describe the mechanism of action of the first classspecific FH inhibitor, 2-thiomalate, through X-ray crystallography and inhibition assays. Our crystal structures of both FH isoforms with inhibitor bound at 2.05 Å resolution and 1.60 Å resolution show high structural similarity. These structures further reveal that the selectivity of 2-thiomalate for class I FHs is due to direct coordination of the inhibitor to the unique Fe of the catalytic [4Fe-4S] cluster that is found in class I parasitic FHs but is absent from class II human FH. These studies provide the structural scaffold in order to exploit class I FHs as potential drug targets against leishmaniases as well as Chagas diseases, sleeping sickness, and malaria. L eishmaniases are neglected tropical diseases (NTDs) caused by the parasite Leishmania spp. There are three clinical forms of leishmaniases: cutaneous, muco-cutaneous, and visceral. Cutaneous, the most common form of the disease, and muco-cutaneous leishmaniases can be caused by L. major, L. panamensis, L. mexicana, L. braziliensis, L. infantum, and L. guyanensis, among other species. The visceral leishmaniasis, the most severe form of the disease, is caused by L. donovani, L. tropica, or L. infantum. Leishmaniases are a major public health problem in low-income countries with approximately 1.3 million new cases and over 20 000 deaths annually, according to the World Health Organization. The control of this disease is challenging due to the growing number of cases in the past 2 decades as a consequence of poverty, climatic and environmental changes, coinfection with HIV, conflict areas, migratory flow, and rapid urbanization. 1,2 A vaccine is not yet available to prevent leishmaniases, and the current treatment is limited to drugs with poor efficacy, high toxicity, high cost, long duration, and/or the requirement of daily injections. 3 Development of resistance to the available drugs is also becoming an issue. 3 Consequently, more effective therapies are needed to fight leishmaniases. Fumarate hydratase (FH; fumarase; EC 4.2.1.2) catalyzes the stereospecific reversible conversion of fumarate to Smalate. This reaction is part of the tricarboxylic acid (TCA) cycle, participates in the succinic fermentation pathway 4 and DNA repair, 5 and is proposed to provide fumarate for the de novo pyrimidine biosynthetic pathway. 6 FHs are grouped in two classes with low amino acid sequence identity (∼20%) and distinct protein structures. 7,8 Class I FHs are [4Fe-4S] clustercontaining dimeric enzymes found in archaea, prokaryotes, and unicellular eukaryotes, including protozoan parasites. 9−11 Class II FHs are iron-independent tetrameric enzymes found in prokaryotes and eukaryotes, including humans. 12 Thus, class I
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Papers by Catherine Drennan