The Old Yellow Enzyme has been shown to catalyze efficiently the NADPH-linked reduction of nitro-olefins. The reduction of the nitro-olefin proceeds in a stepwise fashion, with formation of a nitronate intermediate that is freely dissociable from the enzyme. The first step involves hydride transfer from the enzyme-reduced flavin to carbon 2 of the nitro-olefin. The protonation of the nitronate at carbon 1 to form the final nitroalkane product also is catalyzed by the enzyme and involves Tyr-196 as an active site acid͞base. This residue also is involved in aci-nitro tautomerization of nitroalkanes, the first example of a nonredox reaction catalyzed by the enzyme.O ld Yellow Enzyme (OYE; EC 1.6.99.1), isolated from brewer's bottom yeast, was the first enzyme where the requirement of a prosthetic group (cofactor) for catalysis was demonstrated (1). OYE is a mixture of homodimers and a heterodimer arising from two yeast genes, with each monomer binding a molecule of flavin mononucleotide. Although OYE's exact physiological function is still unknown, many tight binding ligands such as phenols and steroids have been identified, raising the possibility that this enzyme might be involved in yeast sterol metabolism (2). Upon binding with phenols and heteroaromatic compounds containing ionizable hydroxyl groups OYE produces characteristic long wavelength charge transfer complexes (3).The crystal structure of OYE has been solved, revealing several amino acid residues around the active site of the enzyme to effect catalysis and ligand binding (4). Phenolic compounds are held parallel to the flavin by hydrogen bonding of the phenolate oxygen with . The long wavelength absorbance band arises from parallel stacking interaction between the phenolate anion and the flavin. Several mutants of OYE have been constructed and confirmed the importance of , and Thr-37 residues in ligand binding interactions and catalysis (5-7). We also have discovered that this enzyme catalyzes the reduction of the olefinic bond of ␣,␤-unsaturated carbonyl compounds by using NADPH as a reductant (2, 8) and also catalyzes slow aromatization of cyclic enones like cyclohexenone by a novel dismutation reaction (8). Using R-( 2 H) NADPH in D 2 O it was unequivocally established that reduction of ␣,␤-unsaturated carbonyl compounds proceeds via stereospecific transfer of hydride to the carbon atom ␤ to the carbonyl function and transfer a proton to the ␣ position, resulting in trans addition to the double bond (8). Another class of compounds similar to ␣,␤-unsaturated carbonyls is that of unsaturated nitro compounds. Like a carbonyl group a nitro group exerts a strong electron attracting influence within the molecule, enhancing the acidity of the hydrogen atoms attached to the carbon ␣ to the substituent group. Nitro compounds also exhibit a tautomerism exactly analogous to keto-enol tautomerism. Chiral nitro compounds are an important class of synthetically useful compounds, which can be easily converted into chiral amines, and chiral amines are useful interm...

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

Recently there has been a surge of activity in exploring the ability of enzymes to effect chemical transformations with high selectivity. 1 The advent of recombinant DNA technology and sitedirected mutagenesis and the use of nonconventional reaction media like organic solvents has resulted in the effective manipulation of enzymes and in the design of semisynthetic enzymes. 2 The fact that it is relatively easy to remove the flavin prosthetic group to obtain apoprotein and then reconstitute the apoprotein with chemically modified flavins provides flavoproteins with a means of manipulating their catalytic activity. 3 In the present communication we report successful conversion of a flavoenzyme NADPH-dependent reductase to an oxygen-dependent desaturase by this strategy.The R,β-unsaturated ketone or enone functionality enjoys a unique position in organic chemistry as it is involved in a diverse array of reactions such as 1,2-additions or 1,4-conjugate additions, alkylations, or Diels-Alder reactions. It is also a part of numerous natural products. 4 However, the selective catalytic oxidation of simple carbonyl compounds directly to their corresponding R,βunsaturated derivatives (enones) under mild conditions is one of the most difficult reactions to achieve using conventional synthetic methods.Here we present a new general enzymatic catalyst for this transformation, developed by successful manipulation of an enzyme meant for reducing R,β-unsaturated carbonyl compounds to their corresponding saturated derivatives (flavoenzyme reductase) to catalyze exactly the opposite reaction of oxidizing saturated carbonyl compounds to their corresponding R,β-unsaturated enones (desaturase activity). Old yellow enzyme (OYE) is a mixture of homodimers and a heterodimer arising from two yeast genes, with each monomer binding a molecule of flavin mononucleotide (FMN). Although its exact physiological function is still unknown, there is considerable knowledge of structurefunction aspects. The crystal structure is solved, 5 and several tightbinding ligands such as steroids have been identified, raising the * Corresponding author: (tel.

A unique mannose-binding lectin, highly specific for terminal Man(␣1,3)Man groups, was isolated from bulbs of crocus (Crocus vernus All.). The lectin failed to bind to a mannose affinity column and was purified by simple gel permeation chromatography (Sephacryl S200). The purified lectin, obtained in crystalline form, had a molecular mass of 44 kDa on gel filtration and showed a single peptide band with a molecular mass of 11 kDa on SDS-polyacrylamide gel electrophoresis, indicating it to be a tetrameric protein composed of four identical subunits. The N-terminal amino acid sequence analysis of the crocus lectin showed essentially no homology with that of other mannose-binding bulb lectins. The crocus lectin selectively interacted with the wild type Saccharomyces cerevisiae and other mannans carrying terminal Man(␣1,3)Man but not with those lacking this disaccharide unit. In hapten inhibition studies, methyl ␣-mannopyranoside did not inhibit the mannan-lectin interaction. Of various ␣-mannooligosaccharides, those having the Man(␣1,3)Man sequence showed the highest inhibitory potency, confirming the strict requirement of lectin for terminal ␣1,3-linked mannosylmannose units. An affinity column of immobilized lectin enabled the complete resolution of yeast mannan and glycogen. The immobilized lectin may provide a useful tool for purification and analysis of biologically important polysaccharides and glycoproteins.Since the first report on a yeast mannan-binding lectin from bulbs of tulip, Tulipa generiana (1), several kinds of ␣-mannose-binding lectins have been studied in our laboratory, mostly from bulbs of the family Amaryllidaceae, such as Galanthus nivalis (snow drop; GNA) 1 (2, 3), Hippeastrum hybrid (amaryllis), Narcissus pseudonarcissus (daffodil) (4), Sternbergia lutea (5), and Allium sativum (garlic) (6), which belongs to the Lilaceae family. A similar lectin was also isolated from leaves of Listera ovata (twayblade) (7). These lectins are distinct from hitherto known mannose/glucose-binding lectins, such as concanavalin A and other legume lectins, in their strict requirement for the axial C-2 hydroxyl group of ␣-D-mannopyranose. Our detailed studies of the carbohydrate binding specificity of these lectins have indicated some differences with regard to the location of mannosidic linkages at the terminal and/or internal position in the carbohydrate chain. For instance, GNA recognizes terminal Man(␣1,3)Man (3) and also certain internal linkages (8). Similarly, L. ovata lectin can recognize the internal sequence of ␣(1,3)-linked mannosidic linkages (7). In a survey of new plant lectins we found that the bulbs of Crocus vernus All., belonging to the family Iridaceae, accumulates a very unique mannose-binding lectin with a very strict requirement for terminal ␣-1,3-mannosyl mannose units. This lectin, designated CVA, agglutinates rabbit but not human erythrocytes and does not appear to have homology with hitherto known mannose-binding lectins in its sequence of Nterminal amino acids. This paper reports the pu...

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin-Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal α-D-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of α-D-mannopyranosyl and α-D-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.

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