Premium
Site‐directed mutagenesis of selected residues at the active site of aryl‐alcohol oxidase, an H 2 O 2 ‐producing ligninolytic enzyme
Author(s) -
Ferreira Patricia,
RuizDueñas Francisco J.,
Martínez María J.,
van Berkel Willem J. H.,
Martínez Angel T.
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05488.x
Subject(s) - chemistry , active site , site directed mutagenesis , stereochemistry , cofactor , enzyme , biochemistry , oxidase test , residue (chemistry) , mutant , gene
Aryl‐alcohol oxidase provides H 2 O 2 for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl‐alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate‐binding site were chosen for site‐directed mutagenesis modification. Several Pleurotus eryngii aryl‐alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans , and characterized in terms of their steady‐state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl‐alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin‐adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finaly, L315 affects the enzyme k cat , although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl‐alcohol oxidase active site residues.