Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily ...1 and 2 Stig cyclases, which adopt a β‐sandwich fold like the non‐catalytic carbohydrate‐binding motif. Several complex structures were also determined of indole‐based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H‐bond to the indole N and triggers the acid‐catalyzed Cope rearrangement. Through analyzing the enzyme–ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement.
Complex structure elucidations of Stig cyclases reveal a common substrate‐binding mode and mechanism of action of these enzymes. Located near the bottom of a terminal cavity in the β‐sandwich, a strictly conserved aspartate plays a vital role in catalysis, while the surrounding active‐site residues including those in the overhanging loop may determine the type of cyclization and the final product.
We report the first X‐ray structure of the unique “head‐to‐middle” monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl ...diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis‐prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S‐thiolo‐dimethylallyl diphosphate and S‐thiolo‐isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 s a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure‐based mechanism of this unusual prenyl synthase.
Fragrance of lavender: The structure of lavandulyl diphosphate synthase, an irregular monoterpene synthase, was determined and is remarkably similar to the cis‐prenyl synthases involved in bacterial cell wall biosynthesis. Determination of two ligand‐bound structures led to an unusual structure‐based mechanism of action.
Structures of the iridoid synthase nepetalactol synthase in the presence of NAD+, NADPH or NAD+/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can ...be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.
You spin me round: X‐ray structures of iridoid synthase show binding of a transoid substrate, which serves as a model for the catalytic mechanism of progesterone reductase. Formation of the iridoid product requires rotation about C1–C2 to form the cisoid isomer, and rotation about C4–C5 to enable cyclization and lactol production.
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 1.48 Å and 1.66 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.
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•The crystal structures of MoeE5 in complex with NAD+ and sugars were determined.•Substrate disposition in the active site suggests a productive binding mode.•Extensive hydrogen-bond network to UDP-GlcA suggests highly specificity of MoeE5.•The catalytic pathway is elucidated by constructing a product model of UDP-GalA.
Influenza is a contagious acute respiratory disease caused by the influenza virus infection. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the ...influenza virus. Influenza A virus encompasses several different HA subtypes with different strains, which are constantly changing. In this study, we identified a fully human H1N1 neutralizing antibody (32D6) via an Epstein-Barr virus-immortalized B cell-based technology. 32D6 specifically neutralizes the clinically isolated H1N1 strains after the 2009 pandemic but not the earlier strains. The epitope was identified through X-ray crystallographic analysis of the 32D6-Fab/HA1 complex structure, which revealed a unique loop conformation located on the top surface of HA. The major region is composed of two peptide segments (residues 172-177 and 206-213), which form an abreast loop conformation. The residue T262 between the two loops forms a conformational epitope for recognition by 32D6. Three water molecules were observed at the interface of HA and the heavy chain, and they may constitute a stabilizing element for the 32D6-HA association. In addition, each 32D6-Fab is likely capable of blocking one HA trimer. This study provides important information on the strain specificity of 32D6 for the therapeutic treatment and detection of viral infection.
The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista ...and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.
•The structure of N-terminally truncated Nus1 was determined at 2.0 Å resolution.•Significant differences from other cis-prenyltransferase structures were observed.•A model of Nus1/Rer2 complex suggests conserved subunit interface and active site.•The C-terminus of Nus1 interacts with Rer2 but does not contain an RXG motif.•A majority of disease-causing mutation sites in human NgBR can now be located.
Natural orifice transluminal endoscopy has been developed for abdominal surgical procedures. The aim of this study was to compare the surgical outcome between a novel transoral approach and a ...standard transthoracic approach for the thoracic cavity in a canine model.
Twenty-eight dogs were assigned to transoral (n = 14) or standard thoracoscopy (n = 14). Each group underwent thoracic exploration, pre-determined surgical lung biopsy, and pericardial window creation. Blood draws were obtained before surgery and at postoperative days 1, 3, 7, and 14. Operative time, complications, laboratory parameters, hemodynamic parameters, and inflammatory parameters were compared between the two procedures. The animals were monitored for two weeks and necropsy were performed for surgical outcome evaluation.
The thoracic procedures were successfully performed in all of the dogs, with the exception of one animal in the transoral group. There were no serious acute or delayed complications related to surgery. There was no difference between the two surgical groups for each of the hemodynamic parameters that were evaluated. Regarding the immunological impact of the surgeries, transoral thoracoscopy was associated with significant elevations in interleukin 6 and c-reactive protein levels on postoperative days 1 and 3, respectively, when compared with the standard thoracoscopy. All dogs recovered well, without signs of mediastinitis or thoracic infection. Necropsy revealed absence of infection, no injury to vital organs, and confirmed the success of the novel procedure.
This study suggests that both techniques were comparable with respect to procedure success rate, hemodynamic impact, and inflammatory changes. Furthermore, there was no difference in the incidence of postoperative discomfort between groups.
We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an ...AT-rich DNA dodecamer (CGCGAATTCGCG)2 and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100–500 nM, and we found good correlations (R 2 = 0.89, S. aureus; R 2 = 0.79, E. coli) between experimental and predicted cell growth inhibition by using DNA ΔT m and UPPS IC50 experimental results together with one computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multitargeting.
Most known cellulase‐associated carbohydrate‐binding modules (CBMs) are attached to the N‐ or C‐terminus of the enzyme or are expressed separately and assembled into multi‐enzyme complexes (for ...example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR‐220, a GH5‐family endo‐β‐1,4‐glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A‐like catalytic domain with a (β/α)8 TIM‐barrel fold and an inserted CBM29‐like noncatalytic domain with a β‐jelly‐roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full‐length and CBM‐deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full‐length and CBM‐deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single‐chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross‐domain groove. Subsequent site‐specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.
A unique endoglucanase with a carbohydrate‐binding module inserted in the middle of the catalytic domain has been characterized structurally and functionally, providing insights into the mode of action responsible for its enhanced catalytic performance.
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