Publications

• Structural analysis of the catalytic mechanism and stereoselectivity in Streptomyces coelicolor alditol oxidase.

  Forneris F., Heuts D.P., Delvecchio M., Rovida S., Fraaije M.W., Mattevi A.

  Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex ... >> More

  Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity. << Less

  Biochemistry 47:978-985(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472.

  Yamashita M., Omura H., Okamoto E., Furuya Y., Yabuuchi M., Fukahi K., Murooka Y.

  A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel elec ... >> More

  A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)K(m) value for xylitol was two and four times higher than those for galactitol and n-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65 degrees C. The optimal temperature and pH were 55 degrees C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 by that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X-Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications. << Less

  J. Biosci. Bioeng. 89:350-360(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor.

  Heuts D.P., van Hellemond E.W., Janssen D.B., Fraaije M.W.

  A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, ea ... >> More

  A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His(46)) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (K(m) = 0.32 mm, k(cat) = 13 s(-1)) and sorbitol (K(m) = 1.4 mm, k(cat) = 17 s(-1)) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s(-1)), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 x 10(5) m(-1) s(-1)), which confirms that the enzyme represents a true flavoprotein oxidase. << Less

  J. Biol. Chem. 282:20283-20291(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.