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1.
Proc Natl Acad Sci U S A ; 113(28): 7792-7, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27354518

RESUMO

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , DNA/metabolismo , Simulação por Computador , Cristalografia , Escherichia coli , Furanos , Humanos , Modelos Químicos , Timina/análogos & derivados
2.
J Am Chem Soc ; 138(23): 7429-35, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27268299

RESUMO

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.


Assuntos
Lisina/química , Poliubiquitina/química , Poliubiquitina/síntese química , Sítios de Ligação , Cristalografia por Raios X , Endopeptidases/metabolismo , Lisina/metabolismo , Modelos Moleculares , Poliubiquitina/metabolismo , Conformação Proteica , Tioléster Hidrolases/metabolismo , Ubiquitinação
3.
J Am Chem Soc ; 138(43): 14497-14502, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27768314

RESUMO

Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.


Assuntos
Multimerização Proteica , Ubiquitina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , Estereoisomerismo
4.
mLife ; 3(1): 87-100, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38827510

RESUMO

Insertion sequences (ISs) exist widely in bacterial genomes, but their roles in the evolution of bacterial antiphage defense remain to be clarified. Here, we report that, under the pressure of phage infection, the IS1096 transposition of Mycobacterium smegmatis into the lsr2 gene can occur at high frequencies, which endows the mutant mycobacterium with a broad-spectrum antiphage ability. Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes. The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium, thus defending against phage infection. Strikingly, a phage that has evolved mutations in two tail-filament genes can re-escape from the lsr2 inactivation-triggered host defense. This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by IS transposition and provided insight into the natural evolution of bacterial antiphage defense.

5.
Cell Host Microbe ; 31(9): 1469-1480.e4, 2023 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-37567169

RESUMO

In eukaryotic cells, serine/threonine protein kinases (StpKs) play important roles in limiting viral infections. StpKs are commonly activated upon infections, inhibiting the expression of genes central for viral replication. Here, we report that a eukaryotic-like StpK7 encoded by MSMEG_1200 in M. smegmatis is required for mycobacteriophage TM4 to escape bacterial defense. stpK7 is located within a gene island, MSMEG_1191-MSMEG_1200, containing multiple anti-phage genes resembling the BREX (bacteriophage exclusion) phage-resistance system. StpK7 negatively regulates the expression of this gene island. Following phage TM4 infection, StpK7 is induced, directly phosphorylating the transcriptional regulator MSMEG_1198 and inhibiting its positive regulatory activity, thus reducing the expression of multiple downstream genes in the BREX-like gene island. Further analysis showed that genes within this anti-phage island critically regulate mycobacterial lipid hemostasis and phage adsorption. Collectively, this work characterizes a regulatory network driven by StpK7, which is utilized by phage TM4 to escape from the host defense against mycobacteria.


Assuntos
Bacteriófagos , Mycobacterium , Bacteriófagos/genética , Bacteriófagos/metabolismo , Eucariotos , Proteínas Quinases , Células Eucarióticas/metabolismo , Mycobacterium/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Bactérias/metabolismo
6.
J Mol Biol ; 434(13): 167634, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35588869

RESUMO

Ubiquitination, an important posttranslational modification, participates in virtually all aspects of cellular functions and is reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 34 (USP34) plays an essential role in cancer, neurodegenerative diseases, and osteogenesis. Despite its functional importance, how USP34 recognizes ubiquitin and catalyzes deubiquitination remains structurally uncharacterized. Here, we report the crystal structures of the USP34 catalytic domain in free state and after binding with ubiquitin. In the free state, USP34 adopts an inactive conformation, which contains a misaligned catalytic histidine in the triad. Comparison of USP34 structures before and after ubiquitin binding reveals a structural basis for ubiquitin recognition and elucidates a mechanism by which the catalytic triad is realigned. Transition from an open inactive state to a relatively closed active state is coupled to a process by which the "fingertips" of USP34 intimately grip ubiquitin, and this has not been reported before. Our structural and biochemical analyses provide important insights into the catalytic mechanism and ubiquitin recognition of USP34.


Assuntos
Proteases Específicas de Ubiquitina/química , Ubiquitina , Domínio Catalítico , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação
7.
Nat Commun ; 13(1): 4672, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945250

RESUMO

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Oryza , Transdução de Sinais , Animais , Humanos , Mamíferos/metabolismo , Oryza/genética , Oryza/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
8.
Biotechnol Lett ; 33(6): 1107-12, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21279421

RESUMO

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni(2+)-NTA chromatography. Compared with other rPNPs, PNP(816) was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k (cat)/K (m) using inosine as the substrate at 37°C. The PNP(816) or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.


Assuntos
Antivirais/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia , Estabilidade Enzimática , Escherichia coli K12/enzimologia , Escherichia coli K12/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Pseudoalteromonas/enzimologia , Pseudoalteromonas/genética , Nucleosídeos de Purina/biossíntese , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribavirina/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura
9.
Chem Asian J ; 9(8): 2018-29, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24909658

RESUMO

DNA/RNA methylation can be generated by methyltransferases and thus plays a critical role in regulating cellular processes; alternatively, nucleic acid methylation can be produced by methylation agents and is cytotoxic/mutagenic if left unrepaired. Oxidative demethylation mediated by non-heme iron-dependent dioxygenases is an efficient way to reverse either the cellular roles of regulatory methylation or the cytotoxic/mutagenic effects of methylation damage. In this Focus Review we summarize recent advances in the study of nucleic acid dioxygenases exemplified by the TET and AlkB family proteins, with an emphasis on chemical insights from the recent literature. Comparison of the chemical mechanisms of these dioxygenases revealed that differences in the mechanism also contribute significantly to their distinct biological functions.


Assuntos
Metilação de DNA , Dioxigenases/metabolismo , Ferro/metabolismo , RNA/metabolismo , Reparo do DNA , Oxirredução , Especificidade por Substrato
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