Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

Genetic interactions between HNT3/Aprataxin and RAD27/FEN1 suggest parallel pathways for 5′ end processing during base excision repair

Mutations in Aprataxin cause the neurodegenerative syndrome ataxia oculomotor apraxia type 1. Aprataxin catalyzes removal of adenosine monophosphate (AMP) from the 5′ end of a DNA strand, which results from an aborted attempt to ligate a strand break containing a damaged end. To gain insight into which DNA lesions are substrates for Aprataxin action in vivo, we deleted the Saccharomyces cerevisiae HNT3 gene, which encodes the Aprataxin homolog, in combination with known DNA repair genes. While hnt3Δ single mutants were not sensitive to DNA damaging agents, loss of HNT3 caused synergistic sensitivity to H₂O₂ in backgrounds that accumulate strand breaks with blocked termini, including apn1Δ apn2Δ tpp1Δ and ntg1Δ ntg2Δ ogg1Δ. Loss of HNT3 in rad27Δ cells, which are deficient in long-patch base excision repair (LP-BER), resulted in synergistic sensitivity to H₂O₂ and MMS, indicating that Hnt3 and LP-BER provide parallel pathways for processing 5′ AMPs. Loss of HNT3 also increased the sister chromatid exchange frequency. Surprisingly, HNT3 deletion partially rescued H₂O₂ sensitivity in recombination-deficient rad51Δ and rad52Δ cells, suggesting that Hnt3 promotes formation of a repair intermediate that is resolved by recombination.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Atom ordering in cuboctahedral Ni-Al nanoalloys

Energy calculations have been carried out on high-symmetry cuboctahedral Ni-Al nanoalloy clusters, of varying composition, with the interatomic interactions modelled by the Gupta many-body potential. Relaxations of cuboctahedral fragments cut from the bulk lattice of Ni₃Al, with 13-561 atoms, were undertaken, as were relaxations of high symmetry clusters with 55 and 147 atoms. The lowest energy isomers were found to be dominated by three factors: the tendency toward mixing due to the favourable energy of mixing, Δ_mixE; the size difference between nickel and aluminium; and the higher cohesive and surface energy of nickel compared to aluminium. The latter two factors favour Al-segregation to the surface. The most stable Ni:Al composition approaches 3:1 for larger clusters.     

A small one-band paracentric inversion inv (4) (p15.3p16.3).

A neonate with aniridia was found to have a one band paracentric inversion of the short arm of chromosome 4. This was initially difficult to interpret on high resolution banding. The inversion was present in three generations of the family.