Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca(2+) ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca(2+) changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 °C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca(2+) activation, and stabilization.

Afadin, a scaffold protein localized in adherens junctions (AJs), links nectins to the actin cytoskeleton. Nectins are the major cell adhesion molecules of AJs. At the initial stage of cell-cell junction formation, the nectin-afadin interaction plays an indispensable role in AJ biogenesis via recruiting and tethering other components. The afadin PDZ domain (AFPDZ) is responsible for binding the cytoplasmic C-terminus of nectins. AFPDZ is a class II PDZ domain member, which prefers ligands containing a class II PDZ-binding motif, X-U-X-U (U, hydrophobic residues); both nectins and other physiological AFPDZ targets contain this class II motif. Here, we report the first crystal structure of the AFPDZ in complex with the nectin-3 C-terminal peptide containing the class II motif. We engineered the nectin-3 C-terminal peptide and AFPDZ to produce an AFPDZ-nectin-3 fusion protein and succeeded in obtaining crystals of this complex as a dimer. This novel dimer interface was created by forming an antiparallel b sheet between b2 strands. A major structural change compared with the known AFPDZ structures was observed in the a2 helix. We found an approximately 2.5 Å -wider ligand-binding groove, which allows the PDZ to accept bulky class II Abbreviations: AFPDZ, afadin PDZ domain; Bcr, breakpoint cluster region; disc large, zonula occludens-1; PDZ, postsynaptic density-95

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10(6) M(-1) and 10(4) M(-1), respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10(4) M(-1), and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.

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Ishida2, Akinori Ikeuchi2, lzumi Kurnagaii. Mit$uo Umetsui (i7bhoku univ. tech., 2Tbyota Central RdiD tab) Cellulose is naturally occuning plant cell wall constituent and most abundant po]ysaccharides on earth. An economically viable process for converting this biomass to fermentable sugar is a prerequisite for the development ofcellulosic biomass-based bio-refineries for the production of food, feed, chemieals and fuels. Cel]ulDse can be converted to fermentable sugar by microbial cellulose degrading enzyrnes, so ca]]ed ce]lulase. Ce]]u]ases are general]y modular proteins with an independent catalytic domain (CD) and a caTbohydrate-binding module (CBM) and in some bacteria, CDs with different functions are clustered onagiantscaffoldproteincontainingCBM,viacoheisin-dockerininteractionto eencient degradation, so called cellulosome. Recently, we proposcd a new design of artificial cellulosome to enhance ce]]ulase activity with streptavidin or streptavidin modified CdSe quantum dot as a scaffold via biotin-avidin interaction.Activity of the cluster enzyme with two kind of CBD indicated that depend on combination of scafTbld and CBD,

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Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-Lmethionine (SAM) and pyridoxal 5'-phosphate to isomerize L-α-Lys to L-β-Lys. LAM is a member of the radical-SAM enzyme superfamily in which a + cluster reductively cleaves SAM to produce the 5'-deoxyadenosyl radical (5'-dAdo*), which abstracts an H-atom from Lys. 5'-dAdo* is so reactive that it has never been observed, thus this makes characterization of this step difficult. We utilize multinuclear ENDOR to characterize this radical mechanism in LAM by using SAM surrogate. We conclude that the active-site facilitates hydrogen atom transfer by enforcing van der Waals contact between radical and Lys. This constraint enables the enzyme to minimize and even eliminate side reactions. 3P026糖結合モジュール The carbohydrate binding module attached to endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1, CBM-DK, have putative imperfect tandem α-, β-, and γ-repeats. Among the three repeats, we recently showed that the α-repeat mainly contributes to the carbohydrate binding, in which Asp270 and Trp273 have the critical role [1]. While the residues corresponding to Asp 270 are conserved in both β-and γ-repeats, the residues corresponding to Trp273 are Asp314 and Gly358, respectively. In this study, we generated the Trp introduced mutants, D314W and G358W, and analyzed the interactions with laminarin and laminarioligosaccharides, using surface plasmon resonance biosensor and isothermal titration calorimetry.[1] Tamashiro et al., Glycoconj. J. 29, 77-85, 2012. Human carbonic anhydrase II (hCAII) has been extensively studied as a model system for investigating the finely tuned movement of protons in an efficient histidine-regulated hydrogen bond relay during enzymatic catalysis. Here, we report structural information of the histidine (His64), using site-directed mutagenesis and 2D 15N/ 1 H NMR experiments for determining the tautomeric constant of histidine residues. We found that the NMR resonance of 15 N nucleus in the imidzole of His64 splits into two signals in a mutant enzyme. This shows that 1) His64 has two conformations and 2) the chemical exchange is sufficiently slow on the NMR timescale. This feature strongly supports that the proton transfer process does not require a change in orientation of His64 during catalysis. 3P028Rhodococcus rhodochrous Nitrilase is an industrial enzyme that hydrolyzes nitrile compounds into ammonia and carboxylic acids. The nitrilase from Rhodococcus rhodochrous (J1-NTase) associates from an inactive dimer to an active oligomer upon heating. The C-terminus of J1-Ntase is known to be easily degraded by proteases and its loss induces a helical complex with higher activity. We, therefore, examined structural change of J1-Ntase upon heating by 1 H NMR. Despite of its large molecular weight (80kDa in dimer), the methyl signal peak of J1-Ntase was clearly resolved at low temperatures and gradually decreased with temperature increase. We are going to interpret these sepctral changes in comparison with small-angle scattering da...