3P026 Substrate binding ability of the Trp introduced mutant of carbohydrate-binding module(01A. Protein: Structure,Poster)

Abstract

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...