RESUMO
The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.
Assuntos
Antimaláricos/metabolismo , Fosfomicina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Biocatálise , Flavina-Adenina Dinucleotídeo/metabolismo , Fosfomicina/biossíntese , Cinética , Oxigenases de Função Mista/química , Modelos Moleculares , NADP/metabolismo , Domínios ProteicosRESUMO
RNA polymerase inhibitors like the CBR class that target the enzyme's complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg(2+) ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.
Assuntos
Anti-Infecciosos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Nucleotídeos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/metabolismo , Difosfatos/metabolismo , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética/efeitos dos fármacosRESUMO
CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium tuberculosis/enzimologia , RNA Ribossômico/metabolismo , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Modelos Moleculares , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , RNA Ribossômico/genética , VirulênciaRESUMO
Bacteriophage T7 encodes an essential inhibitor of the Escherichia coli host RNA polymerase (RNAP), the product of gene 2 (Gp2). We determined a series of X-ray crystal structures of E. coli RNAP holoenzyme with or without Gp2. The results define the structure and location of the RNAP σ(70) subunit domain 1.1(σ(1.1)(70)) inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the open promoter complex. The structures and associated data, combined with previous results, allow for a complete delineation of the mechanism for Gp2 inhibition of E. coli RNAP. In the primary inhibition mechanism, Gp2 forms a protein-protein interaction with σ(1.1)(70), preventing the normal egress of σ(1.1)(70) from the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP holoenzyme, σ(1.1)(70), to inhibit the function of the enzyme.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Escherichia coli/virologia , Modelos Moleculares , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/farmacologia , Fator sigma/antagonistas & inibidores , Cristalografia por Raios X , Estrutura Terciária de Proteína/efeitos dos fármacosRESUMO
Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.
Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Bacilos Gram-Positivos Formadores de Endosporo/enzimologia , Trissacarídeos/química , Xilanos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Genoma Bacteriano/fisiologia , Bacilos Gram-Positivos Formadores de Endosporo/genética , Hidrólise , Família Multigênica/fisiologia , Via de Pentose Fosfato/fisiologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Terciária de Proteína , Trissacarídeos/metabolismo , Xilanos/metabolismoRESUMO
Phosphonate natural products possess a range of biological activities as a consequence of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The dianionic form of these compounds at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates. Microorganisms producing phosphonates have evolved two solutions to overcome this hurdle: biosynthesis of monoanionic phosphinates containing two P-C bonds or esterification of the phosphonate group. The latter solution was first discovered for the antibiotic dehydrophos that contains a methyl ester of a phosphonodehydroalanine group. We report here the expression, purification, substrate scope, and structure of the O-methyltransferase from the dehydrophos biosynthetic gene cluster. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-hydroxyethylphosphonate, 1,2-dihydroxyethylphosphonate, and acetyl-1-aminoethylphosphonate. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the antimalaria clinical candidate fosmidomycin, showing its promise for applications in bioengineering.
Assuntos
Vias Biossintéticas/fisiologia , Metiltransferases/química , Metiltransferases/metabolismo , Organofosfonatos/metabolismo , Streptomyces/enzimologia , Bioengenharia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cristalografia por Raios X , Dipeptídeos/biossíntese , Esterificação , Fosfomicina/análogos & derivados , Fosfomicina/metabolismo , Cinética , Espectrometria de Massas , Metilação , Estrutura Molecular , S-Adenosilmetionina/metabolismo , Especificidade por SubstratoRESUMO
The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 Å resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.
Assuntos
Acetiltransferases/química , Proteínas de Bactérias/química , Dobramento de Proteína , Streptomyces/enzimologia , Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Fosfomicina/análogos & derivados , Fosfomicina/biossíntese , Fosfomicina/química , Organofosfonatos , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B(1)4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a ß-D-fucosidase. We also present the 2.2 Å resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respective active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have ß-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.
Assuntos
Glicosídeo Hidrolases/química , alfa-L-Fucosidase/química , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Modelos Moleculares , Prevotella/enzimologia , Especificidade por SubstratoRESUMO
Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridium/metabolismo , Glucose/metabolismo , Lectinas/metabolismo , Manose/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridium/química , Clostridium/genética , Evolução Molecular , Glucose/química , Glucose/genética , Lectinas/química , Lectinas/genética , Ligantes , Manose/química , Manose/genética , Mutação de Sentido Incorreto , Ligação Proteica , Especificidade por Substrato , Thermoanaerobacterium/química , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismoRESUMO
The minichromosome maintenance (MCM) proteins, members of the AAA+ (ATPase associated with diverse cellular activities) superfamily, are believed to constitute the replicative helicase in eukaryotic and archaeal species. Here, we present the 1.9 A resolution crystal structure of a monomeric MCM homolog from Methanopyrus kandleri, the first crystallographic structure of a full-length MCM. We also present an 18 A cryo-electron microscopy reconstruction of the hexameric MCM from Methanothermobacter thermautotrophicus, and fit the atomic resolution crystal structure into the reconstruction in order to generate an atomic model for the oligomeric assembly. These structural data reveal a distinct active site topology consisting of a unique arrangement of critical determinants. The structures also provide a molecular framework for understanding the functional contributions of trans-acting elements that facilitate intersubunit crosstalk in response to DNA binding and ATP hydrolysis.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Sequência de Aminoácidos , Animais , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Domínio Catalítico , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Células Eucarióticas/enzimologia , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Movimento , Regiões Promotoras Genéticas/genética , Bactérias/enzimologia , Domínio Catalítico , Cristalização , DNA/química , DNA/metabolismo , Genes Reporter , Cinética , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Rotação , Eletricidade Estática , Moldes GenéticosRESUMO
We report here the enzymatic biosynthesis of FR-900098 analogues and establish an in vivo platform for the biosynthesis of an N-propionyl derivative FR-900098P. FR-900098P is found to be a significantly more potent inhibitor of Plasmodium falciparum 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PfDxr) than the parent compound, and thus a more promising antimalarial drug candidate.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Antimaláricos/farmacologia , Desenho de Fármacos , Fosfomicina/análogos & derivados , Plasmodium falciparum/enzimologia , Aldose-Cetose Isomerases/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Fosfomicina/química , Fosfomicina/farmacologia , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacosRESUMO
A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Transativadores/química , Transativadores/metabolismo , Thermus/química , Thermus/enzimologiaRESUMO
Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Regiões Promotoras Genéticas , Cristalografia por Raios X , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Thermus/química , Thermus/enzimologiaRESUMO
L-arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD(+) reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 A resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP(+) as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.
Assuntos
Neurospora crassa/enzimologia , Desidrogenase do Álcool de Açúcar/química , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Conformação Proteica , Engenharia de Proteínas , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/metabolismo , Álcoois Açúcares/metabolismoRESUMO
Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward beta-1,4-linked sugars, they can engage both cello- and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.
Assuntos
Proteínas de Bactérias/química , Metabolismo dos Carboidratos , Thermoanaerobacterium/enzimologia , Sequência de Aminoácidos , Calorimetria , Biologia Computacional , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Especificidade por Substrato , TermodinâmicaRESUMO
Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.