Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design.
D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of k(cat) with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k(cat)/K(M) is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D-galactose. Batch conversion experiments of D-galactose to 2-keto-D-galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics.
Department of Food Sciences and Technology, BOKU, Vienna, Austria.
Clara
C
Salaheddin
Oliver
O
Spadiut
Roland
R
Ludwig
Tien-Chye
TC
Tan
Christina
C
Divne
Dietmar
D
Haltrich
Clemens
C
Peterbauer
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D019149:Bioreactors; D002021:Buffers; D002237:Carbohydrate Dehydrogenases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D055454:Trametes / Q000201:enzymology* / Q000235:genetics
D002021:Buffers; D010710:Phosphates; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005690:Galactose