Many bacteria are able to grow with L-arabinose as the sole carbon and energy source, and the bacterial pathway of L-arabinose metabolism has been extensively investigated. Many bacteria including Escherichia coli depend on protein products of the araBAD operon, which contains araB (ribulokinase, EC 2.7.1.16), araA (L-arabinose isomerase, EC 5.3.1.4) and araD (L-ribulose-phosphate 4-epimerase, EC 5.1.3.4) to convert L-arabinose to D-xylulose 5-phosphate through L-ribulose and L-ribulose 5-phosphate (1). In a recently characterized fungal pathway (2-4), L-arabinose is also converted to D-xylulose 5-phosphate but through different intermediates by two reductases, two dehydrogenases, and a kinase. On the other hand, it is believed that a hypothetical pathway of L-arabinose metabolism is operative in some bacteria (5-13) (Fig. 1A). In this pathway, L-arabinose is oxidized to L-arabino-␥-lactone by NAD(P) ϩ -dependent dehydrogenase. The lactone is cleaved by a lactonase to L-arabonate, followed by two successive dehydration reactions forming L-2-keto-3-deoxyarabonate (L-KDA) 2 and ␣-ketoglutaricsemialdehyde(␣KGSA).ThelaststepistheNAD(P) ϩ -dependent dehydrogenation of ␣KGSA to ␣-ketoglutaric acid (referred to as the first pathway). Alternatively, L-KDA is cleaved through an aldolase reaction to glycolaldehyde and pyruvate (referred to as the second pathway).

We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.Keywords: coiled coil; de novo design; folding; helical structures; metalloproteins Studies on de novo designed proteins involve the construction of proteins with unique tertiary structures and the creation of new functional proteins. The coiled coil structure is often observed in natural proteins for the intermolecular assemblies of the functional domains. This motif, due to its structural simplicity and biomolecular significance, has been the subject of extensive analyses to understand the principles of de novo design, as well as protein folding and stability~Lau et al., 1984;O'Neil & DeGrado, 1990;Harbury et al., 1993!. Furthermore, the designed coiled coils can be fused to various functional peptides or domains of natural proteins for biological and medical applications~Pack & Plückthun, 1992; Hodges, 1996; Terskikh et al., 1997!.The designed coiled coil, which drastically changes its conformation depending on external stimuli, should be useful to control the associations and the functions of domains attached to the peptide. Among the various external stimuli, metal binding has been studied extensively, and the factors required for metal binding are well understood. A variety of metal binding sites have been designed, but they are on the surfaces of preformed artificial supramolecules, such as an a-helical bundle protein~Handel & Regan & Clarke, 1990;Regan, 1995;Dieckmann et...

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We have demonstrated recently that nitrous acid or nitric oxide converts 2'-deoxyguanosine (dGuo) into 2'-deoxyoxanosine (dOxo) [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., & Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In the present study, we have measured susceptibility of the N-glycosidic bond of dOxo to spontaneous hydrolysis and its base-pairing stability to evaluate the biological significance of dOxo as a new lesion in DNA. When oligodeoxynucleotide d(T5OT6) (O = dOxo), isolated from nitrous acid-treated d(T5GT6), was incubated at pH 4.0 and 70 degrees C, hydrolysis of the N-glycosidic bond of dOxo occurred with a first-order rate constant. Comparison of the rate constants with those of dGuo and dXao indicates that the N-glycosidic bond of dOxo was as stable as that of dGuo in d(T5GT6) and hydrolyzed 44-fold more slowly than that of 2'-deoxyxanthosine (dXao), a simultaneously generated damage by nitrous acid and nitric oxide. For the estimation of the base-pairing stability, UV melting curves were measured for the duplexes of d(T5OT6).d(A6NA5) (N = A, G, C, and T) at neutral pH. The Tm values obtained were 15.3, 14.1, 19.3, and 16. 3 degrees C for N = A, G, C, and T, respectively, which are much lower than that of the intact duplex containing a G.C pair at the same position [d(T5GT6).d(A6CA5), Tm = 32.8 degrees C] but comparable with those of d(T5XT6).d(A6NA5) (X = dXao, Tm = 14.8-22.3 degrees C). CD spectra of the four duplexes containing dOxo showed preservation of the structure of the intact duplex at low temperature. UV and NMR pH-titration studies indicated the pKa for the ring-opening and -closing equilibrium to be 9.4, implying that dOxo is in the ring-closed form at physiological pH. This structure appears to be not suitable geometrically for the hydrogen bond formation with a specific counter base, thus causing equally low Tm values for all the counter bases. Consequently, these results imply that dOxo, a novel DNA lesion, may have an important and unique role in mutagenic events in cells.