Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor.
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.
Heuts
Dominic P H M
DP
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
van Hellemond
Erik W
EW
Janssen
Dick B
DB
Fraaije
Marco W
MW
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics; D014993:Xylitol / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D005420:Flavoproteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C515743:alditol oxidase (FAD), Streptomyces coelicolor; D014993:Xylitol