Unspecific peroxygenases
(UPOs) are glycosylated fungal enzymes
that can selectively oxidize C–H bonds. UPOs employ hydrogen
peroxide as the oxygen donor and reductant. With such an easy-to-handle
cosubstrate and without the need for a reducing agent, UPOs are emerging
as convenient oxidative biocatalysts. Here, an unspecific peroxygenase
from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six
putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate
selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions,
such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations
were demonstrated for the nonenantioselective oxidation of racemic
1-phenylethanol rac-1b (TON = 13 000),
giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated
yield. HspUPO features a compact and rigid three-dimensional
conformation that wraps around the heme and defines a funnel-shaped
tunnel that leads to the heme iron from the protein surface. The tunnel
extends along a distance of about 12 Å with a fairly constant
diameter in its innermost segment. Its surface comprises both hydrophobic
and hydrophilic groups for dealing with substrates of variable polarities.
The structural investigation of several protein–ligand complexes
revealed that the active site of HspUPO is accessible
to molecules of varying bulkiness with minimal or no conformational
changes, explaining the relatively broad substrate scope of the enzyme.
With its convenient expression system, robust operational properties,
relatively small size, well-defined structural features, and diverse
reaction scope, HspUPO is an exploitable candidate
for peroxygenase-based biocatalysis.
Cardiac senescence
is a typical chronic frailty condition in the
elderly population, and cellular aging is often associated with oxidative
stress. The mitochondrial-membrane flavoenzyme monoamine oxidase A
(MAO A) catalyzes the oxidative deamination of neurotransmitters,
and its expression increases in aged hearts. We produced recombinant
human MAO A variants at Lys305 that play a key role in O
2
reactivity leading to H
2
O
2
production. The
K305Q variant is as active as the wild-type enzyme, whereas K305M
and K305S have 200-fold and 100-fold lower
k
cat
values and similar
K
m
. Under
anaerobic conditions, K305M MAO A was normally reduced by substrate,
whereas reoxidation by O
2
was much slower but could be
accomplished by quinone electron acceptors. When overexpressed in
cardiomyoblasts by adenoviral vectors, the K305M variant showed enzymatic
turnover similar to that of the wild-type but displayed decreased
ROS levels and senescence markers. These results might translate into
pharmacological treatments as MAO inhibitors may attenuate cardiomyocytes
aging.
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