Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

The present work addresses the investigation of the influence of substitution on the initial spin state in photoinduced electron-transfer (PET) reactions with a series of four exciplex systems i.e., N-ethylcarbazole (ECZ)−1,4-dicyanobenzene (DCB), 1,4,5,8,9-pentamethylcarbazole (PMC)−DCB, ECZ−1,2,4,5-tetracyanobenzene (TCNB), and PMC−TCNB by means of a low magnetic field (MF) (0.05 T). The two primary intermediates that play major roles in determining the efficiencies of bimolecular PET reactions are the contact ion pair (CIP), i.e., (A-D+), and the solvent-separated ion pair (SSIP) (A-(S)D•+). The effect of MF of the order of hyperfine interaction present in the system on such reactions reflects the unique combination of spin dynamics, diffusion dynamics, and geminate recombination in the SSIPs. Thus MF can be successfully used to investigate the initial spin state of a SSIP where electronic coupling between acceptor (A) and donor (D) molecules is small indeed. The experimental techniques have used either laser flash photolysis to estimate the magnetic field effect (MFE) on triplet free ions or an improved phase-sensitive detection system to measure the enhancement in singlet CIP or exciplex luminescence. By the changes of the substituents in A/D molecules, the modifications in the production of either singlet or triplet SSIPs have been discussed. The observed MFEs have been correlated with the Marcus relation between free energy changes and redox potentials. Another novel finding is that MFE on exciplex luminescence is controlled not only by the dielectric of the medium but the extent of electronic coupling, i.e., the extent of charge transfer (δ), between D and A molecules also plays a major role in it. The deviation in εmax, the dielectric for maximum MFE, from the previously obtained values has been discussed on the basis of the modification in the potential energy surfaces between CIP and SSIP, which has been further supported by an analytical model.

A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity. In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 x 10(-4) U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies.

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The electron transfer (ET) process between a fluorophore (N-ethylcarbazole) and a quencher (1,6dicyanobenzene) has been studied by absorption, steady-state and time-resolved fluorescence and laser flash photolysis techniques in homogeneous and micellar media. The effect of a magnetic field on the ET process of this system has also been carried out by flash photolysis. The results reflect the following unique features of this system. The reduction potentials of the acceptor and the donor are such that the exciplex formation is possible by ET, moreover, as the charge separation is small a weak exciplex is formed even in a highly polar solvent, e g . acetonitrile (MeCN). Thus, the exciplex formation and solvent-separated ion pair (SSIP) formation in MeCN take place simultaneously. The experimentally obtained bimolecular quenching constant, k, , from the Stern-Volmer plot, agrees satisfactorily with theoretically calculated k, for an ET reaction applying Marcus theory. The exciplex formation inside the micellar medium enables us to study the magnetic field effect (MFE) on this exciplex system. The simultaneous occurrence of the exciplex and radical ion pair in MeCN as well as the formation of the exciplex within the micellar environment, most probably for the first time, ensure a special characteristic of this exciplex system. Another novel feature is the abnormal behaviour of this system in benzene, which has also been discussed here.