A novel Acetivibrio cellulolyticus anchoring scaffoldin that bears divergent cohesins

Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed. The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here. The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two approximately 30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface.     

The 0.93A crystal structure of sphericase: a calcium-loaded serine protease from Bacillus sphaericus

We have previously isolated sphericase (Sph), an extracellular mesophilic serine protease produced by Bacillus sphaericus. The Sph amino acid sequence is highly homologous to two cold-adapted subtilisins from Antarctic bacilli S39 and S41 (76% and 74% identity, respectively). Sph is calcium-dependent, 310 amino acid residues long and has optimal activity at pH 10.0. S41 and S39 have not as yet been structurally analysed.In the present work, we determined the crystal structure of Sph by the Eu/multiwavelength anomalous diffraction method. The structure was extended to 0.93A resolution and refined to a crystallographic R-factor of 9.7%. The final model included all 310 amino acid residues, one disulfide bond, 679 water molecules and five calcium ions. Although Sph is a mesophilic subtilisin, its amino acid sequence is similar to that of the psychrophilic subtilisins, which suggests that the crystal structure of these subtilisins is very similar.The presence of five calcium ions bound to a subtilisin molecule, as found here for Sph, has not been reported for the subtilisin superfamily. None of these calcium-binding sites correlates with the well-known high-affinity calcium-binding site (site I or site A), and only one site has been described previously. This calcium-binding pattern suggests that a reduction in the flexibility of the surface loops of Sph by calcium binding may be responsible for its adaptation to mesophilic organisms.     

Biotransformations of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked mutants

Propenylbenzenes are often used as starting materials in the chemical synthesis of aroma compounds and fine chemicals. In the present study, we demonstrate the ability of an Arthrobacter sp. to transform various structures of propenylbenzenes derived from essential oils to flavor, fragrance, and fine chemicals. Arthrobacter strain TA13 and its t-anethole blocked mutants (incapable of growing on t-anethole) converted isoeugenol to vanillin and vanillic acid; and safrole to hydroxychavicol. High conversion efficiencies were achieved in the biotransformations of isosafrole to piperonylic acid, and eugenol to a mixture of ferulic acid and vanillic acid. In addition, anisic acid was produced in high yields from t-anethole, anisyl alcohol, or anisaldehyde. The accumulation of the corresponding aromatic acids from the tested propenylbenzenes is due to the lack of m-demethylase activity in strain TA13 that prevents further cleavage of the benzene ring. Interestingly, in the transformation of eugenol (a 2-propenylbenzene) the side chain was initially oxidized to the corresponding cinamic acid derivative (ferulic acid) while the 1-propenylbenzenes gave substituted benzoic acids, suggesting two different chain shortening mechanisms.     

Crystal structure and snapshots along the reaction pathway of a family 51 alpha-L-arabinofuranosidase

High-resolution crystal structures of alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (beta/alpha)8-barrel and a 12-stranded beta sandwich with jelly-roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl-enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 A apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion-like transition state via charge delocalization and specific protein-substrate interactions. The enzyme is an anti-protonator, and a 1.7 A electrophilic migration of the anomeric carbon takes place during the hydrolysis.     

Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus

Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Br?nsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.     

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.     

Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Br?nsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Absorption of CIS Immigrants into Israeli Schools: A Semipermeable Enclave Model

National ideology, institutional arrangements, and immigrant community self-image should be taken into account in order to understand the way immigrant students insert themselves into their host society. We found that immigrant students from the CIS (the former Soviet Union) adopted a "semipermeable enclave" mode of integration into the Israeli education system in the 1990s. CIS immigrants' high self-esteem along with the schools' so-called "pluralistic ideology" and ineffective arrangements to apply the official assimilationist national ideology produced this new absorption pattern. Because immigrant children's integration into schools is a dynamic and dialectic process resulting from the interaction between immigrants and the host society, its analysis must take into consideration not only educational policies, but also social conditions such as power relations between different ethnic groups, as well as the characteristics of the specific immigrant community.