Human indoleamine 2,3-dioxygenase (IDO) catalyzes the cleavage of the pyrrol ring of L-Trp and incorporates both atoms of a molecule of oxygen (O 2). Here we report on the x-ray crystal structure of human IDO, complexed with the ligand inhibitor 4-phenylimidazole and cyanide. The overall structure of IDO shows two ␣-helical domains with the heme between them. A264 of the flexible loop in the heme distal side is in close proximity to the iron. A mutant analysis shows that none of the polar amino acid residues in the distal heme pocket are essential for activity, suggesting that, unlike the heme-containing monooxygenases (i.e., peroxidase and cytochrome P450), no protein group of IDO is essential in dioxygen activation or proton abstraction. These characteristics of the IDO structure provide support for a reaction mechanism involving the abstraction of a proton from the substrate by iron-bound dioxygen. Inactive mutants (F226A, F227A, and R231A) retain substratebinding affinity, and an electron density map reveals that 2-(Ncyclohexylamino)ethane sulfonic acid is bound to these residues, mimicking the substrate. These findings suggest that strict shape complementarities between the indole ring of the substrate and the protein side chains are required, not for binding, but, rather, to permit the interaction between the substrate and iron-bound dioxygen in the first step of the reaction. This study provides the structural basis for a heme-containing dioxygenase mechanism, a missing piece in our understanding of heme chemistry.iron ͉ kynurenine ͉ tryptophan ͉ x-ray crystallography O xygenases (1) are metal-containing enzymes that catalyze the incorporation of a molecule of oxygen (O 2 ) into the substrate, and thus play a crucial role in the metabolism and synthesis of a variety of biological substances. Two types of oxygenase are currently known: monooxygenases (scheme I) and dioxygenases (scheme II):In the 1950s and 1960s, Hayaishi and coworkers reported that two heme-containing dioxygenases, indoleamine 2,3-dioxygenase (IDO) (2) and tryptophan 2,3-dioxygenase (TDO) (3), catalyze the initial and rate-limiting step of L-Trp catabolism in the kynurenine (Kyn) pathway (4). This step involves the oxidative cleavage of the 2,3 double bond in the indole moiety of L-Trp, resulting in the production of N-formyl Kyn. Increased levels of the Kyn pathway metabolites quinolinic acid and 3-hydroxykynurenine (3OHKyn) have been observed in a number of neurological or psychiatric disorders. L-Trp-derived UV filters (Kyn and 3OHKyn glucoside) can bind to the lens protein and appear to be mainly responsible for the nuclear cataract (5). L-Trp also serves as a precursor for the synthesis of the neurotransmitter serotonin and the hormone melatonin. IDO exhibits a broader substrate specificity than TDO, because the former can degrade indoleamines, including L-Trp, D-Trp, serotonin, melatonin, and tryptamine (6). In addition to its role as a L-Trp-catabolizing enzyme, IDO is involved in the immunoregulating system [review by Mellor and M...

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The stability of red radish extract to light, heat, and hydrogen peroxide at different pH values (3, 5, and 7) was examined, in which major anthocyanins were pelargonidin glycosides acylated with a combination of p-coumaric, ferulic, or caffeic acids. The light irradiation (fluorescence light, 5000 lx; at 25 degrees C) indicated that the red radish extract was more stable at lower pH than at higher pH. The HPLC analyses revealed that diacylated anthocyanins in the extract were more stable to light at pH 3 than monoacylated anthocyanins. No significant difference in degradation rates of acylated anthocyanins at pH 5 was observed, whereas anthocyanins acylated with p-coumaric or ferulic acids were more stable at pH 7 than ones with caffeic acids. The stability to heat (at 90-95 degrees C) showed a tendency similar to that for light. The number of intramolecular acyl units contributes to stability to light and heat at lower pH, whereas the characteristics of intramolecular acyl units influence the stability at higher pH. The degradation behavior of red radish extract to H2O2 were almost the same to those of light and heat, depending on the pH. However, HPLC analyses revealed that the stability of individual acylated anthocyanins were independent of the pH. These data suggest that the characteristics, the number, and the binding site of intramolecular acyl units affect the stability of anthocyanin to H2O2. DPPH radical scavenging activity of all acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside. The activity of acylated anthocyanins mostly depended on the activity of intramolecular acyl units (caffeic acid > ferulic acid > p-coumaric acid). However, the activity was highly affected by the binding site of intramolecular acyl units even if anthocyanins have common acyl units.

Steady-state-kinetics investigations were carried out for the oxidation of aldose sugars by soluble quinoprotein glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus using N-methylphenazonium methyl sulfate (PMS) as artificial electron acceptor. As is not uncommon for a dye-linked dehydrogenase, the enzyme showed ping-pong behaviour and double-substrate inhibition. However, under conditions that avoided its masking by sugar-substrate inhibition as much as possible, negative kinetic cooperativity with respect to sugar substrate oxidation by this enzyme was demonstrated. Arguments are presented that exclude trivial factors as a cause for the phenomenon observed. Experimental data could be fitted with an equation accounting for biphasic cooperativity containing two sets of apparent kinetic parameters, V 1 and K 1 , and V 2 and K 2 , representing the enzyme's Michaelis-Menten behaviour at low and high substrate concentrations, respectively. Assuming that subunit interaction causes the cooperativity effect, the sets express the performance of soluble GDH's two subunits in two states of mutual interaction. From fitting the experimental data for several sugars with this equation, it appeared that their V 1 values were similar, although their K 1 values varied considerably. This showed that the cooperativity effect dramatically changes the performance of soluble GDH, as reflected by the V 2 and K 2 values for glucose (in phosphate buffer) being about 10-fold and 100-fold higher than the V 1 and K1 values, respectively. Substituting the Ca 2ϩ involved in activation of pyrroloquinoline quinone (PQQ) in soluble GDH by Sr 2ϩ affected the cooperativity effect (an increase of the K 2 value) but not the two turnover rates of the hybrid enzyme for glucose. The data suggest that the two catalytic cycles of soluble GDH have different ratelimiting steps compared with that of PQQ-containing methanol dehydrogenase.Keywords : quinoprotein ; glucose dehydrogenase ; enzyme kinetics; cooperativity.The bacterium Acinetobacter calcoaceticus produces two active hybrid holoenzyme can be achieved by adding PQQ and Cd 2ϩ , Mn 2ϩ , or Sr 2ϩ . Hybrid enzymes prepared with Cd 2ϩ or pyrroloquinoline quinone (PQQ)-containing glucose dehydrogenases (GDH), a soluble one and a membrane-bound one. The Mn 2ϩ are nearly as active as normal holo-enzyme (in the standard assay with 50 mM glucose) but enzyme prepared with Sr 2ϩ homodimeric soluble GDH has been purified from this bacterium in the holoform [1Ϫ5] and in the apoform from an overpro-showed only half of the activity. The reason for this suboptimal performance is unknown. ducing Escherichia coli recombinant strain [6]. The enzyme has a broad specificity with respect to aldose sugars (hexoses, As isolated or as obtained after reconstitution of the apoenzyme with PQQ holoGDH contains PQQ in the oxidized form pentoses, monosaccharides, and disaccharides) and artificial electron acceptors {two-electron [e.g. N-methylphenazonium only [3, 6]. On addition of glucose, reduction of the cofactor tak...