Biochemical characterization of mutants in the active site residues of the beta-galactosidase enzyme of Bacillus circulans ATCC 31382

The Bacillus circulans ATCC 31382 beta-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost) all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447) and nucleophile (E532), and as transition state stabilizers (H345, H379), respectively. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.</p

Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae

β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with (13)C6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site.</p

Metal ion responsive adhesion of vesicles by conformational switching of a non-covalent linker

This contribution describes the metal ion responsive adhesion of vesicles induced by a conformational switch of a non-covalent linker molecule. A p-tert-butylbenzyl dimer with a flexible N,N'-bis(3-aminopropyl)ethylenediamine spacer was used as a non-covalent linker, which induces aggregation and adhesion (but not fusion) of host bilayer vesicles composed of amphiphilic beta-cyclodextrins by the formation of hydrophobic inclusion complexes. The aggregation and adhesion of the vesicles in dilute aqueous solution was confirmed by isothermal titration calorimetry (ITC), optical density measurements at 600 nm (OD600), dynamic light scattering (DLS), zeta-potential measurements, cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy. However, in the presence of a divalent metal ion like Cu(2+), the tetra-amine linker molecule forms a stable metal coordination complex and dramatically switches its conformation from linear to bent, which results in the dissociation of intervesicular complexes, and leads to the dispersion of vesicle clusters. This process is reversible in the presence of a strong metal ion chelator, such as EDTA, that scavenges the Cu(2+) ion complexed by the linker. The linker molecule regains its linear conformation and triggers the reaggregation of the vesicles. In contrast, conformational switching was inhibited by introducing a rigid N,N'-bis(3-aminopropyl)piperazine spacer in the non-covalent linker molecule and vesicles do not aggregate in the presence of a cyclic guest that can only bind intravesicularly. Thus, a metal ion regulated molecular switch can control the aggregation state of an organic colloidal solution.</p

The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane

The mannitol permease (EIIMtl) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EIIMtl contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EIIMtl, all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EIIMtl undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Vipp1 and PspA: Related but not twins

The Vesicle Inducing Protein in Plastids 1 (Vipp1) was suggested to be involved in thylakoid membrane formation in both chloroplasts and cyanobacteria. The protein shows sequence homology to the Phage Shock Protein A (PspA) from bacteria, and both proteins have similar secondary structures. 2D-structures of PspA and of Vipp1 have been determined by electron microscopy in the recent years. Both PspA and Vipp1 form large homooligomeric rings with high molecular masses but their ring dimensions differ significantly. Furthermore, Vipp1 forms rings with different rotational symmetries whereas PspA appears to form rings with singular rotational symmetry. In this article addendum we compare the structures of PspA and Vipp1. Furthermore, we suggest a spatial structural model of the observed Vipp1 rings