The SARS coronavirus main proteinase (M(pro)) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by X-ray crystallography to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the M(pro) by analysis of multiple crystal structures (including two new crystal forms) and by molecular dynamics (MD) calculations. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymatic activity of the M(pro) monomer and the need for dimerization are also discussed.

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the “SARS-unique domain”. The X domain was proposed to be an ADP-ribose-1″-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro ) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CL pro s by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CLpro , the N-terminal deleted SARS 3CL pro still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (K d ) of the N-terminal deleted proteinase dimer (262 M) is very similar to that of the full-length proteinase dimer (227 M). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CL pro . Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.

EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that methylates lysine 27 of histone 3. Overexpression of EZH2 has been found in a wide range of cancers, including those of the prostate and breast. In this review, we address the current understanding of the oncogenic role of EZH2, including its PRC2-dependent transcriptional repression and PRC2-independent gene activation. We also discuss the connections between EZH2 and other silencing enzymes, such as DNA methyltransferase and histone deacetylase. We comprehensively address the architecture of the PRC2 complex and the crucial roles of each subunit. Finally, we summarize new progress in developing EZH2 inhibitors, which could be a new epigenetic therapy for cancers.

We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3C pro ). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3C pro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3C pro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic ␣,␤-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3C pro , which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1=; the most potent inhibitors comprise P4 to P1=. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC 50 ] ‫؍‬ 0.5 M for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC 50 s of Ϸ180 nM against EV71 and Ϸ60 nM against human rhinovirus 14 in a live virus-cell-based assay. Even the shorter SG75, spanning only P3 to P1=, displayed significant activity (EC 50 ‫؍‬ 2 to 5 M) against various rhinoviruses.

A highly efficient and green process for the hydrogenation of biomass‐derived levulinic acid (LA) to γ‐valerolactone (GVL) has been developed. GVL was obtained in a yield of 99.9 mol % with a turnover frequency as high as 7676 h−1 in aqueous medium by using a Ru/TiO2 catalyst under mild reaction conditions (70 °C). The strong interaction between Ru and TiO2 facilitated both the dispersion of Ru nanoparticles and the stability of the catalyst. In addition, as solvent, water participated in the hydrogenation of LA, which was confirmed by an isotope‐ labeling experiment with D (D2O). Specifically, the H atom(s) in water took part in the hydrogenation of the CO group of LA, which promoted the catalytic activity and GVL yield remarkably.

The relationships of extant and extinct lineages of Adephaga were analysed formally for the first time. Emphasis is placed on the aquatic and semiaquatic groups and their evolution in the Mesozoic. †Triadogyrus and †Mesodineutus belong to Gyrinidae, the sister group of the remaining families. †Triaplidae are the sister group of the following groups (Haliplidae, Geadephaga, Dytiscoidea incl. †Liadytidae, †Parahygrobiidae and †Coptoclavidae [major part]). The lack of a ventral procoxal joint and a very short prosternal process are plesiomorphies of †Triaplidae. †Coptoclavidae and †Timarchopsinae are paraphyletic. †Timarchopsis is placed in a geadephagan clade. In contrast to other coptoclavids, its metathorax is close to the condition found in Haliplidae, with a complete transverse ridge and coxae with large plates and free mesal walls. †Coptoclavidae s.str., i.e. excl. †Timarchopsis, is a dytiscoid subgroup. The mesal metacoxal walls are fused, the coxal plates are reduced, and the transverse ridge is absent. †Stygeonectes belongs to this dytiscoid coptoclavid unit and is therefore misplaced in †Timarchopsinae. †Liadytidae belongs to a dytiscoid subgroup, which also comprises the extant families Aspidytidae, Amphizoidae, Hygrobiidae and Dytiscidae. †Parahygrobia is the sister group of Hygrobiidae. The larvae are characterized by a broad gula, the absence of the lacinia, retractile maxillary bases and very long urogomphi set with long setae. †Liadytiscinae is the sister group of extant Dytiscidae. There is no support for a clade †Eodromeinae and for Trachypachidae incl. †Eodromeinae. †Fortiseode is nested within Carabidae. The exclusion of fossil taxa has no effect on the branching pattern. The evolution of Adephaga in the Mesozoic is discussed. Possible reasons for the extinction of †Coptoclavidae are the rise of teleost fish and the competition of Gyrinidae and Dytiscidae, which possess efficient defensive glands and larval mandibular sucking channels.