Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis.
Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.
Madzak
C
C
Laboratoire de Synthèse Sélective Organique et Produits Naturels, UMR CNRS 7573, ENSCP, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France.
Mimmi
M C
MC
Caminade
E
E
Brault
A
A
Baumberger
S
S
Briozzo
P
P
Mougin
C
C
Jolivalt
C
C
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D004271:DNA, Fungal / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics
D000814:Aniline Compounds; D004271:DNA, Fungal; D011994:Recombinant Proteins; C016835:2,5-xylidene; D042845:Laccase