An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.This work is supported by the Danish Council for Independent Research|Natural Sciences (FNU) [grant number 09-072151], by 1/3 PhD fellowship from the Technical University of Denmark (to CW) and by a Hans Christian Ørsted postdoctoral fellowship from DTU (to DC).This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00253-016-7417-

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Screening for galectin-3 inhibitors from synthetic lacto-N-biose libraries using microscale affinity chromatography coupled to mass spectrometry

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Natural and recombinant A and B gene encoded glycosyltransferases

NRC publication: Ye

Mapping the active site of the bacterial enzyme LpxC using novel carbohydrate-based hydroxamic acid inhibitors

LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase), an enzyme involved in the biosynthesis of lipid A, is crucial for the growth of Gram-negative bacteria. This enzyme has accordingly been identified as a potential target for the development of novel antibiotics against Gram-negative bacteria. The carbohydrate-derived hydroxamic acid 1 (1,5-anhydro-2-C- (carboxymethyl N-hydroxyamide)-2-deoxy-3-O-myristoyl-D-glucitol) was previously shown to exhibit a wide spectrum of inhibitory activity against LpxC enzymes. Here we describe the preparation of seven analogs of 1 and their enzymatic evaluation. Two of the hydroxyl groups (OH-3 and 6) of the GlcNAc residue were found to be involved in the binding interaction, and there is an important hydrophobic interaction in the vicinity O-3 position with the enzyme that recognizes aromatic as well as aliphatic substituents. Copyright © Taylor & Francis, Inc.link_to_subscribed_fulltex

Synthesis of a carbohydrate-derived hydroxamic acid inhibitor of the bacterial enzyme (LpxC) involved in lipid A biosynthesis

(Matrix presented) The enzyme LpxC (UDP-3-O-[(R)-3-hydroxymyristoyl] -GlcNAc deacetylase) catalyzes the second step of lipid A biosynthesis and is essential for bacterial growth. A GlcNAc-derived hydroxamic acid inhibitor 8 of this enzyme was synthesized using two different routes. Compound 8 exhibits activity toward LpxC enzymes from a wider spectrum of bacterial species than any of the previously reported hydroxamic acid inhibitors.link_to_subscribed_fulltex

Assignment of the 1H, 13C and 15N resonances of the LpxC deacetylase from Aquifex aeolicus in complex with the substrate-analog inhibitor TU-514 [6]

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Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide

Peer reviewed: YesNRC publication: Ye