Biology Reference
In-Depth Information
et al., 1995
) and in the non-lignolytic fungus Penicillium simplicissimum
(
de Jong et al., 1992
).
AAOs are produced by a small number of fungal species, and their physi-
ological role has so far been hardly studied compared with the other fungal
oxidoreductases. The AAO of P. chrysosporium is produced in nitrogen-rich
media during the primary metabolism. This enzyme is also the only AAO
reported as being inducible by aryl-alcohols such as vanillyl alcohol. The
maximum production of AAO by P. chrysosporium is obtained at the end of
growth when glucose becomes limiting (
Asada et al., 1995
). Pleurotus AAOs
are the most thoroughly studied AAOs. The best production of a natural
AAO (2500 U/L) was obtained during culture of P. pulmonarius in a medium
containing glucose and peptones (
Varela et al., 2000a
). An AAO from
P. eryngii was cloned and expressed in A. nidulans. This heterologous expres-
sion allowed the production of 400-500 U/L, approximately 10 times more
than the production of AAO by P. eryngii cultivated in optimal conditions
(
Varela et al., 2001
).
AAOs are flavoproteins containing one FAD group per enzyme molecule.
They catalyse the oxidation of
a
-and
b
-unsaturated aryl-
g
-alcohols to their
corresponding aldehydes, with the concomitant reduction of O
2
to H
2
O
2
(
Muheim et al., 1990
). They are glycoproteins with molecular masses ranging
from 70 to 80 kDa. Two isoenzymes of AAO were identified in P. sajor-caju on
the basis of a difference in pI, which was confirmed by the N-terminal analysis
of these proteins. The N-terminus sequence of P. ostreatus and P. eryngii are
very conserved and contain consensus sequences Gly-Xaa-Gly-Xaa-Xaa-Gly,
characteristic of dinucleotide binding proteins (
Wierenga et al., 1985
).
After the cloning of the gene coding for the AAO of P. eryngii and the
crystallization of the protein, the tri-dimensional structure of this enzyme
was determined. This AAO is apparently organized in 15 helices and two
b
-sheet domains. This enzyme may also be stabilized by a disulfide bridge
between cysteines 248 and 263 (
Varela et al., 2000b
). The crystallization of
the P. pulmonarius AAO gave some further information. The N-terminus
region of this enzyme presents a
b
-
a
-
b
motif containing three glycine residues
involved in the non-covalent binding of FAD (residues 25-60). Glycine 530
and proline 537 may be involved in the active site. However, resolution at
2.8
˚
failed to give more detailed information on the structure (
Varela et al.,
2000a
). FAD acts as a redox centre in the transfer of two hydrogen atoms
and two electrons from aryl-alcohols to dioxygen. Its characteristic absor-
bance at 465 nm is useful to follow the protein during purification steps
(
Varela et al., 2000a
). Studies of the catalytic capacities of AAOs showed
that they all have a wide specificity for various methoxy-substituted phenols
(Bourbonnais and Paice, 1988;
Guillen et al., 1990; Sannia et al., 1991
).
