Distortion of mannoimidazole supports a B 2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens

Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2-B2,5-1S5 conformational itinerary for enzymes of this family

YihQ is a sulfoquinovosidase that cleaves sulfoquinovosyl diacylglyceride sulfolipids

Sulfoquinovose is produced by photosynthetic organisms at a rate of 1010 tons per annum and is degraded by bacteria as a source of carbon and sulfur. We have identified Escherichia coli YihQ as the first dedicated sulfoquinovosidase and the gateway enzyme to sulfoglycolytic pathways. Structural and mutagenesis studies unveiled the sequence signatures for binding the distinguishing sulfonate residue and revealed that sulfoquinovoside degradation is widespread across the tree of life

Interplay between COVID-19, pollution, and weather features on changes in the incidence of acute coronary syndromes in early 2020

Coronavirus disease 2019 (COVID-19) has caused an unprecedented change in the apparent epidemiology of acute coronary syndromes (ACS). However, the interplay between this disease, changes in pollution, climate, and aversion to activation of emergency medical services represents a challenging conundrum. We aimed at appraising the impact of COVID-19, weather, and environment features on the occurrence of ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI) in a large Italian region and metropolitan area

An epoxide intermediate in glycosidase catalysis

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism

Dissecting conformational contributions to glycosidase catalysis and inhibition

Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochemistry at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the critical points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallography of enzymes bound to ligands that mimic the critical points, along with advanced computational methods and kinetic isotope effects are discussed

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet

Development of molecular tools to provide mechanistic insights into diverse glycosidases

© 2016 Dr. Gaetano SpecialeGlycoside hydrolases are enzymes that catalyze the scission of the glycosidic bond. Despite the simplicity of the reaction they exist in a remarkable variety, numbering more than two hundred thousand known gene sequences grouped into more than 130 sequence related families. Understanding the unique features of each enzyme family is a key strategy for their utilization in biotechnology and for rational approaches to the treatment of diseases that involve glycoside hydrolases. The first part of this thesis involves the study of enzymes of glycoside hydrolase family 99 (GH99), a family of endo-α-1,2-mannosidases/endo-α-1,2-mannanases that have been proposed to undertake catalysis through a 1,2-anhydro sugar intermediate. A set of isotopically-labelled mannosides is synthesized and used for the measurements of kinetic isotope effects (KIEs) for basic solvolysis of 4-nitrophenyl α-D-mannopyranoside, which is known to proceed through a 1,2-anhydro sugar. The measured kinetic isotope effect (KIE) for H1, H2, C1, C2, O1 and O2 are 1.112, 1.045, 1.026, 0.999, 1.040, and 1.044, respectively. The transition state for the hydrolysis reaction was modeled with ab initio theoretical methods using the experimental KIE values as constraints. The results are consistent with the reaction mechanism involving rate-limiting formation of a transient 1,2-anhydro sugar that opens in water to give α-D-mannopyranose. These studies provide insight into the nature of the transition state of a bonafide neighboring group participation involving a 1,2-anhydro sugar and benchmark data for interpreting equivalent KIE data for the family GH99 mechanism. Next, a series of unlabelled and five-isotopically-labelled tetrasaccharides are synthesized and used to gain insight in the mechanism of a bacterial GH99 with a combination of protein crystallography, computational modelling and KIE measurements. The X-ray complex between a tetrasaccharide and a catalytically-inactive acid/base mutant enzyme shows an undistorted 4C1 conformation in the –1 subsite of the ground-state Michaelis complex. Using this complex as an input, computational modelling of the enzymic reaction leading to product revealed a 4C1 ↔ [4E]‡ ↔ 4H5 conformational itinerary. The measured H1-KIE value for α-2H was 1.134 (± 0.009), suggesting a dissociative, early TS. These data are collectively consistent with the proposed neighboring group participation mechanism. Compelling evidence for or against this mechanism, with an interpretation supported by the benchmark data for the chemical solvolysis process, should be available when KIE measurements for the enzyme-catalyzed reaction are complete. The second part of the thesis involves the study of the poorly characterized endo-α-1,6-mannosidases of family GH76, and their involvement in the degradation of dietary fungal α-mannan. Using substrates and inhibitors synthesized for the purpose, a comprehensive study of the mechanism of these enzymes is undertaken, supported by site-directed mutagenesis, X-ray crystallography and computational free energy landscape analysis. This work reveals that enzymes of this family operate through a classical Koshland retaining mechanism and follow a typical mannosidase conformational itinerary of OS2 ↔ [B2,5]‡ ↔ 1S5, with the peculiarity of an hydrogen bond of the substrate 3-OH to the incoming/departing nucleophilic carboxylate. The final part of the thesis reports the discovery and characterization of the first dedicated sulfoquinovosidase, YihQ from E. coli, a member of glycoside hydrolase family GH31. Sulfoquinovosidase activity is necessary for the liberation of the sulfonated sugar sulfoquinovose from the sulfolipid sulfoquinovoside diacylglycerol, thus releasing sulfoquinovose for catabolism through the sulfoglycolysis pathway. Synthesis of the chromogenic substrate 4-nitrophenyl α-D-sulfoquinovoside and its use in kinetic assays allowed demonstration that YihQ is retaining glycosidase and the first dedicated α-sulfoquinovosidase. Kinetic and structural analysis of a series of enzyme mutants led to identification the catalytic residues and a triad of amino acids important for sulfonate binding and catalysis. Sequence analysis revealed YihQ orthologs to be widespread in bacteria, fungi, protozoa, metazoan and plants and thus an important gateway enzyme to the catabolic arc of the biogeochemical sulfur cycle

A Facile Synthesis of α-N-Ribosyl-Asparagine and α-N-Ribosyl-Glutamine Building Blocks

Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring post-translational modification of proteins at nucleophilic side chain of amino acid residues. Elucidation of ADP-ribosylation events would benefit greatly from the availability of well-defined ADP-ribosylated peptides and analogues thereof. In this paper we present a novel approach to the chemical synthesis of ribosylated amino acid building blocks using traceless Staudinger ligation. We describe an efficient and stereoselective synthesis of α-N-ribosyl-asparagine (α-N-ribosyl-Asn) and α-N-ribosyl-glutamine (α-N-ribosyl-Gln) building blocks starting from 5-tert-butyldiphenylsilyl-β-d-ribofuranosyl azide. The N-glycosyl aminoacids are produced in good yields as pure α-anomers, suitably protected for peptide synthesis

Safety and outcome of rheolytic thrombectomy for the treatment of acute massive pulmonary embolism

Percutaneous rheolytic thrombectomy is an attractive alternative to thrombolytic therapy in patients with acute pulmonary embolism (PE), but its use is currently discouraged due to safety concerns.Background: Percutaneous rheolytic thrombectomy is an attractive alternative to thrombolytic therapy in patients with acute pulmonary embolism (PE), but its use is currently discouraged due to safety concerns. Methods: We studied 33 consecutive patients (age, 43 ± 13 years; 20 men and 13 women) with acute PE and contraindications to thrombolytic therapy who had rheolytic thrombectomy with the AngioJet catheter (Boston Scientific). Acute massive PE was initially diagnosed by computed tomography and then confirmed by pulmonary angiography. Pulmonary thrombus location was evaluated prior to the procedure. Anemia was defined as a decrease in hematocrit level <39% for men and <36% for women. Renal failure was defined as oliguria (urine output <500 mL/24 hours) or an increase in creatinine (>25% over baseline or an overall increase by 1 g/dL). Results: Catheter thrombectomy resulted in angiographic improvement in 32 patients (96%), with a rapid amelioration in functional class (from 3.3 ± 0.9 to 2.1 ± 0.7; P<.001) and an increase in oxygen saturation (from 71 ± 15% to 92 ± 17%; P<.001). No patient died. Side effects included transient heart block (n = 1), hypotension (n = 3), and bradycardia (n = 5). Anemia occurred in 4 patients, while renal failure was not detected. Clinical improvement was maintained during follow-up. At 1 year, systolic pulmonary pressure was significantly lower than at baseline (65 ± 31 mm Hg vs 31 ± 19 mm Hg; P<.001). Conclusions: Catheter thrombectomy with AngioJet in patients with acute massive PE and contraindications to thrombolysis is an effective therapeutic alternative that is not associated with relevant and persistent side effects, including the risk of death or developing anemia and renal failure