l-amino acid oxidases (LAAOs, EC 1.4.3.2) are oxidoreductases, which catalyze the oxidative deamination of l-amino acids to imino acids. Due to spontaneous hydrolysis, the corresponding α-keto acid and ammonia are formed. As a byproduct, hydrogen peroxide is formed during regeneration of the non-covalently bound cofactor flavin adenine dinucleotide (FAD; Pollegioni, Motta, & Molla, 2013). LAAOs are found in several organisms like mammals, bacteria, algae, and fungi even though the functions differ in different organisms. Snake venom LAAOs (SV-LAAO) are the best-characterized enzymes and can cause apoptosis, edema, or hemolysis (
l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.
l
‐amino acid oxidases (LAAOs) catalyze the oxidative deamination of
l
‐amino acids to corresponding α‐keto acids. Here, we describe the heterologous expression of four fungal LAAOs in
Pichia pastoris
.
cg
LAAO1 from
Colletotrichum gloeosporioides
and
nc
LAAO1 from
Neurospora crassa
were able to convert substrates not recognized by recombinant 9His‐
hc
LAAO4 from the fungus
Hebeloma cylindrosporum
described earlier thereby broadening the substrate spectrum for potential applications. 9His‐
fr
LAAO1 from
Fibroporia radiculosa
and 9His‐
la
LAAO2 from
Laccaria amethystine
were obtained only in low amounts. All four enzymes were N‐glycosylated. We generated mutants of 9His‐
hc
LAAO4 lacking N‐glycosylation sites to further understand the effects of N‐glycosylation. All four predicted N‐glycosylation sites were glycosylated in 9His‐
hc
LAAO4 expressed in
P
.
pastoris
. Enzymatic activity was similar for fully glycosylated 9His‐
hc
LAAO4 and variants without one or all N‐glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N‐glycans. This was caused by the absence of a hypermannosylated N‐glycan on asparagine residue N54. The lack of one or all of the other N‐glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N‐glycosylation during biosynthesis.
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