Search

Mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) are identified in familial Parkinson’s disease. However, it remains unclear whether the phosphorylated Ub ...

MINDY-1 is a recently discovered new family of deubiquitinating enzymes (DUB), but one of its yeast homologs, YGL082W, does not show any DUB activity in vitro. Sequence alignment shows that YGL082W possesses the correct catalytic triad, and yet did not catalyze either the hydrolysis of di-ubiquitin, crosslinking with C-terminally propargylated ubiquitin, or hydrolysis of ubiquitin-7-amino-4-methylcoumarin. After obtaining a crystal structure of the catalytic domain of YGL082W, we identified an interesting difference between the catalytic center loop of YGL082W and that of its human homolog MINDY-1. Because the conformation of the catalytic center loop was previously reported to be important for the deubiquitination activity of MINDY-1, we hypothesized that Glu27 (instead of the corresponding Pro136 in MINDY-1) of the catalytic center loop of YGL082W may impair the conformational change and account for the lack of activity. This hypothesis was supported by homology modeling and molecular dynamics simulations, which showed that the Pro-to-Glu mutation (P136E mutation for MINDY-1) creates a hydrogen bond that inhibits the conformation change of the catalytic center loop of MINDY-1. Further experiments through site-directed mutation validated this hypothesis, showing that the P27E mutation caused MIY1 (a homologous active DUB from yeast) to lose activity.

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.

Met1 type ubiquitination and deubiquitination are involved in the regulation of many fundamental processes such as inflammation and innate immunity, and their interference by pathogens can suppress immune responses in human cells. However, no plant-derived deubiquitinases (DUBs) against Met1 ubiquitin chains have been reported. Using a dehydroalanine (DHA)-bearing Met1 diubiquitin (Met1-diUb) suicide probe, synthesized in one-pot, we identified OTUB1 from Oryza sativa (OsOTUB1) and uncovered its preference for Met1 ubiquitin chains. Also, by resolving the apo structure of OsOTUB1 and its complex with Ub or Met1-diUb, we demonstrated that OsOTUB1 hydrolyses Met1 ubiquitin chains by activation of both the distal and proximal ubiquitin, which is different from OTULIN and expands our mechanistic understanding of the DUB-mediated hydrolysis of Met1 ubiquitin chains. Through large-scale sequence alignment and hydrolysis experiments, two sites in the S1' pocket of the OTUB subfamily (OTUBs) were found to determine the hydrolytic ability of OTUBs against Met1 ubiquitin chains, regardless of species. Furthermore, by analyzing the species distribution of OTUBs capable of hydrolyzing Met1 ubiquitin chains, we found that whereas this activity does not exist in metazoans, it is conserved in green plants (Viridiplantae). This discovery may inform studies of the differentiation between primitive plants and animals.

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.

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.

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.

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.

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.

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 Ni2+-NTA chromatography. Compared with other rPNPs, PNP816 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 PNP816 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.