RESUMEN
Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S-adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.
Asunto(s)
Conformación de Ácido Nucleico , ARN Catalítico , ARN , ARN Catalítico/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , Metilación , ARN/metabolismo , ARN/genética , ARN/química , Humanos , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Nucleótidos/metabolismo , Nucleótidos/química , Nucleótidos/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/química , Especificidad por Sustrato , Animales , Modelos MolecularesRESUMEN
Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient ß1-loop-α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding ß1-loop-α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.
Asunto(s)
Coenzimas , NAD , NAD/metabolismo , Proteínas/química , NADP/metabolismo , S-AdenosilmetioninaRESUMEN
Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Bryopsida , Quitosano , Lipooxigenasa/genética , Quitosano/farmacología , NAD , Bryopsida/genética , Peroxidasas/genética , Peroxidasa/genética , Peroxidasa/metabolismo , Plantas/metabolismoRESUMEN
Life on Earth is driven by electron transfer reactions catalyzed by a suite of enzymes that comprise the superfamily of oxidoreductases (Enzyme Classification EC1). Most modern oxidoreductases are complex in their structure and chemistry and must have evolved from a small set of ancient folds. Ancient oxidoreductases from the Archean Eon between ca. 3.5 and 2.5 billion years ago have been long extinct, making it challenging to retrace evolution by sequence-based phylogeny or ancestral sequence reconstruction. However, three-dimensional topologies of proteins change more slowly than sequences. Using comparative structure and sequence profile-profile alignments, we quantify the similarity between proximal cofactor-binding folds and show that they are derived from a common ancestor. We discovered that two recurring folds were central to the origin of metabolism: ferredoxin and Rossmann-like folds. In turn, these two folds likely shared a common ancestor that, through duplication, recruitment, and diversification, evolved to facilitate electron transfer and catalysis at a very early stage in the origin of metabolism.
Asunto(s)
Transporte de Electrón , Evolución Molecular , Oxidorreductasas/metabolismo , Ferredoxinas/metabolismo , Flavodoxina/metabolismo , Conformación ProteicaRESUMEN
Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.
Asunto(s)
Acetilglucosamina/metabolismo , Bacillus subtilis/enzimología , Glicósido Hidrolasas/metabolismo , Ácidos Murámicos/metabolismo , Peptidoglicano/metabolismo , Cristalografía por Rayos X , Glicósido Hidrolasas/química , Hidrólisis , Conformación ProteicaRESUMEN
Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic ß2 and ß7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.
Asunto(s)
Péptido Sintasas/química , Péptido Sintasas/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Burkholderiaceae/efectos de los fármacos , Burkholderiaceae/genética , Cromatografía en Gel , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Porphyromonas gingivalis is a bacterial species known to be involved in the pathogenesis of chronic periodontitis, that more recently has been as well associated with Alzheimer's disease. P. gingivalis expresses a glutaminyl cyclase (PgQC) whose human ortholog is known to participate in the beta amyloid peptide metabolism. We have elucidated the crystal structure of PgQC at 1.95 Å resolution in unbound and in inhibitor-complexed forms. The structural characterization of PgQC confirmed that PgQC displays a mammalian fold rather than a bacterial fold. Our biochemical characterization indicates that PgQC uses a mammalian-like catalytic mechanism enabled by the residues Asp149, Glu182, Asp183, Asp218, Asp267 and His299. In addition, we could observe that a non-conserved Trp193 may drive differences in the binding affinity of ligands which might be useful for drug development. With a screening of a small molecule library, we have identified a benzimidazole derivative rendering PgQC inhibition in the low micromolar range that might be amenable for further medicinal chemistry development.
Asunto(s)
Aminoaciltransferasas/química , Porphyromonas gingivalis/enzimología , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Bencimidazoles/química , Bencimidazoles/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos MolecularesRESUMEN
BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.
Asunto(s)
Pirroles/metabolismo , Zymomonas/metabolismo , Catálisis , Electrones , Cinética , NAD/metabolismo , NADP/metabolismo , Oxidorreductasas/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD+ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD+/UDP-GlcA and MoeE5/NAD+/UDP-glucose, determined at 1.48â¯Å and 1.66â¯Å resolution. The cofactor NAD+ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD+, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.
Asunto(s)
Antibacterianos/biosíntesis , Oligosacáridos/biosíntesis , UDPglucosa 4-Epimerasa/metabolismo , Antibacterianos/química , Cristalografía por Rayos X , Modelos Moleculares , Oligosacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimología , Especificidad por Sustrato , UDPglucosa 4-Epimerasa/química , UDPglucosa 4-Epimerasa/genéticaRESUMEN
Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/enzimología , Disulfuros/química , Flavina-Adenina Dinucleótido/química , Oxidorreductasas/química , Synechocystis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Membrana Celular/química , Membrana Celular/enzimología , Cristalografía por Rayos X , Cianobacterias/genética , Disulfuros/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Synechocystis/genética , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
Asunto(s)
Péptido Sintasas/química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , Relación Estructura-ActividadRESUMEN
Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination.
Asunto(s)
Prolina/química , Pirrolina Carboxilato Reductasas/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Cinética , Ligandos , Mutación , NADP/química , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Ultracentrifugación , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
FAD and NAD(P) together represent an ideal pair for coupled redox reactions in their capacity to accept two electrons and their redox potentials. Enzymes that bind both NAD(P) and FAD represent large superfamilies that fulfill essential roles in numerous metabolic pathways. Adrenodoxin reductase (AdxR) shares Rossmann fold features with some of these superfamilies but remains in a group of its own in the absence of sequence homology. This article documents the phylogenetic distribution of AdxR by examining whole genome databases for Metazoa, Plantae, Fungi, and Protista, and determines the conserved structural features of AdxR. Scanning these databases showed that most organisms have a single gene coding for an AdxR ortholog. The sequence identity between AdxR orthologs is correlated with the phylogenetic distance among metazoan species. The NADP binding site of all AdxR orthologs showed a modified Rossmann fold motif with a GxGxxA consensus instead of the classical GxGxxG at the edge of the first ßα-fold. To examine the hypothesis that enzyme-coenzyme interfaces represent the conserved regions of AdxR, the residues interfacing FAD and NADP were identified and compared with multiple-sequence alignment results. Most conserved residues were indeed found at sites that surround the interfacing residues between the enzyme and the two coenzymes. In contrast to protein-protein interaction hot-spots that may appear in isolated patches, in AdxR the conserved regions show strict preservation of the overall structure. This structure maintains the precise positioning of the two coenzymes for optimal electron transfer between NADP and FAD without electron leakage to other acceptors.
Asunto(s)
Ferredoxina-NADP Reductasa/química , Ferredoxina-NADP Reductasa/genética , Ferredoxina-NADP Reductasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Coenzimas/metabolismo , Secuencia Conservada/genética , Transporte de Electrón , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/genética , Flavina-Adenina Dinucleótido/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , NADP/química , NADP/genética , NADP/metabolismo , Filogenia , Alineación de SecuenciaRESUMEN
The recently discovered cytokinin (CK)-specific phosphoribohydrolase "Lonely Guy" (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function.
Asunto(s)
Aminohidrolasas/química , Carboxiliasas/química , Claviceps/enzimología , Proteínas Fúngicas/química , Modelos Moleculares , Secuencia de Aminoácidos , Aminohidrolasas/metabolismo , Carboxiliasas/metabolismo , Citocininas/metabolismo , Proteínas Fúngicas/metabolismoRESUMEN
Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity.
Asunto(s)
Corynebacterium/enzimología , Liasas/química , Liasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Epiclorhidrina/metabolismo , Liasas/genética , Modelos Moleculares , Nitrilos/química , Nitrilos/metabolismo , Propanoles/química , Propanoles/metabolismo , Conformación Proteica , EstereoisomerismoRESUMEN
The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Thermoanaerobacter/metabolismo , Cristalografía por Rayos X , NAD/metabolismo , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
3-Ketosteroid Δ(1)-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ(1)-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ(1)-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr(119), Tyr(318), Tyr(487), and Gly(491). Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr(318), respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr(487) and the backbone amide of Gly(491). Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr(487) and Gly(491) work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr(119), the general base Tyr(318) abstracts the axial ß-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Oxidorreductasas/química , Oxidorreductasas/genética , Rhodococcus/enzimología , Rhodococcus/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase catalyzes the interconversion of UDP-GlcNAc to UDP-N-acetylmannosamine (UDP-ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP-GlcNAc 2-epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP-GlcNAc binding, the enzyme undergoes conformational changes involving a rigid-body movement of the C-terminal domain. We also present the crystal structure of Bacillus subtilis UDP-GlcNAc 2-epimerase in the closed conformation in the presence of UDP and UDP-GlcNAc. Although a structural overlay of these two closed-form structures reveals that the substrate-binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP-GlcNAc 2-epimerases. This is the first report on the crystal structure of archaeal UDP-GlcNAc 2-epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme.
Asunto(s)
Proteínas Arqueales , Methanocaldococcus/enzimología , Uridina Difosfato N-Acetilglucosamina , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Isomerismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.