The pseudoalteromonas luteoviolacea L-amino acid oxidase with antimicrobial activity is a flavoenzyme

Article abstract of DOI:10.3390/md16120499

The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.

Full text of DOI:10.3390/md16120499

marine drugs
Article
The Pseudoalteromonas luteoviolacea L-amino Acid
Oxidase with Antimicrobial Activity
Is a Flavoenzyme
Andrés Andreo-Vidal 1, Antonio Sanchez-Amat 1,
*
and Jonatan C. Campillo-Brocal 1,2,
*
1
Department of Genetics and Microbiology, University of Murcia, 30100 Murcia, Spain; andres.andreo@um.es
Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry,
Biotechnology and Health (CBH), KTH Royal Institute of Technology, AlbaNova University Center,
SE-106 91 Stockholm, Sweden
2
*
Correspondence: antonio@um.es (A.S.-A.); jonatancristian.campillo@um.es (J.C.C.-B.);
Tel.: +34-868-884-955 (A.S.-A.); +34-606-634-475 (J.C.C.-B.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 15 November 2018; Accepted: 7 December 2018; Published: 12 December 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The marine environment is a rich source of antimicrobial compounds with promising
pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of
the highest proportions of bacterial species producing antimicrobial molecules. For decades,
the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in
Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification,
cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both
LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a
broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible
for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned
or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing
LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of
the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao
similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides
insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and
shows an effective approach for the detection of novel LAAOs, whose study may be useful for
biotechnological applications.
Keywords: L-amino acid oxidase; antimicrobial activity; flavin cofactor; Pseudoalteromonas luteoviolacea
1. Introduction
Marine ecological niches are excellent sources for many bioactive compounds of biotechnological
and pharmaceutical interest. Among them, antimicrobial compounds synthetized by marine organisms
offer a promising alternative to antibiotics [
well-known producers of several metabolites with antimicrobial activity, such as alkaloids, polyketides,
peptides, and proteins [ ]. The genus Pseudoalteromonas includes Gram-negative, heterotrophic, and
1]. Microorganisms from the Pseudoalteromonas genus are
2,3
aerobic marine bacteria, which belong to the Alteromonadales order in the Gammaproteobacteria class.
They are commonly found in association with seawater macroorganisms, where they play a critical
role in holobiont homeostasis through their metabolic activities [2]. Pseudoalteromonas luteoviolacea
was described in 1976, and was reported to produce antibacterial polyanionic substances inhibited
by catalase.
L-Amino acid oxidases (LAAOs) are enzymes that catalyze the oxidative deamination of amino
acids, releasing the corresponding keto acid, ammonium, and hydrogen peroxide. Two groups of
Mar. Drugs 2018, 16, 499; doi:10.3390/md16120499
www.mdpi.com/journal/marinedrugs

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these enzymes have been described so far. The best-known group (EC 1.4.3.2) utilizes flavin adenine
dinucleotide (FAD) as a cofactor, and oxidizes amino acids in the alpha position (Figure 1). The second
group has recently been described as containing a quinone cofactor generated by post-translational
modification of the protein [
named as LodA-like proteins, after the description of LodA, a L-lysine epsilon-oxidase [
4
]. This group possesses a distinct evolutionary origin [
5
] and has been
]. LAAOs
6
are distributed in many biological groups including bacteria, fungi, algae, plants, insects, molluscs,
fishes, and mammals (including humans), although the most studied members of this group are the
LAAOs present in snake venom [
antimicrobial properties that have been related to different physiological processes, such as the
biocontrol agents in fungi against microbial competitors [ ] or protection of fish skin and gills from
7,8]. The generation of hydrogen peroxide gives to these enzymes
9
bacterial infections [10]. LAAOs are of great biotechnological interest in different applications, such
as the design of biosensors, biotransformations, or biomedicine, and are attracting more and more
interest because of their broad and relevant biological functions [7,11].
Figure 1. Reaction catalyzed by flavin adenine dinucleotide (FAD)-dependent L-amino acid
oxidases (LAAOs).
LAAOs from marine bacteria are believed to play important biological and ecological roles in
their niches, mediated by antimicrobial activity. This is the case of the L-lysine
gammaproteobacteria Marinomonas mediterranea (LodA) and Pseudoalteromonas tunicata (AlpP), which
are involved in biofilm development and dispersal through hydrogen peroxide generation [ 12].
ε-oxidases from the
1,
Some other LAAOs with an antimicrobial activity have been described and characterized in different
marine bacteria. PfaP from Pseudoalteromonas flavipulchra JG1 shares a high similarity with AlpP and
LodA [13]. P. flavipulchra C2 was reported to have a LAAO of a broad substrate range, oxidizing L-Lys,
L-Met, L-Glu, L-Leu, L-Gln, L-Tyr and L-Phe, which contained a nine amino-acid fragment similar
to AlpP [14]. P. luteoviolacea produces a protein with an L-amino acid oxidase (LAAO) activity of
a broad-substrate spectrum, in which the hydrogen peroxide generated mediates its antimicrobial
activity, although the gene coding for this enzyme has not been identified [15,16]. Aquimarina sp.
antisso-27 also synthesizes a broad spectrum LAAO (L-Leu > L-Ile > L-Met > L-Val), with both
antibacterial and algicidal activity [17]. It is worth mentioning that all of these antimicrobial LAAOs
are synthesized by bacteria associated with the microbiota of higher marine organisms, and none of
them have been described with a flavin cofactor [5].
Recently, our group has reported the presence of PlGoxA, a LAAO very specific for Gly synthetized
by P. luteoviolacea CPMOR-2 [18]. PlGoxA contains a cysteine tryptophylquinone (CTQ) cofactor
generated by the post-translational modification of two residues in the same protein. This protein
bears similarity to GoxA from M. mediterranea [19]. Regardless, PlGoxA did not show a broad substrate
range, and thus the LAAO with an antimicrobial activity described in P. luteoviolacea CPMOR-2
still remains to be determined [16]. In the present study, we show that the antimicrobial activity of
Pseudoalteromonas luteoviolacea is due to an L-amino acid oxidase with a flavin cofactor. We believe that
our findings provide insights into the biological and ecological significance of antimicrobial LAAOs in
the Pseudoalteromonas genus.

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2. Results
2.1. Identification of the Gene Encoding the LAAO Activity in CPMOR-2 Strain
As reported by Gómez et al. (2008), Pseudoalteromonas luteoviolacea CPMOR-2 synthesizes a protein
with an antimicrobial and amino acid oxidase activity, although the gene encoding this protein had
not been identified. With this purpose, first, we investigated the optimal conditions for the expression
of LAAO activity. Hence, P. luteoviolacea CPMOR-2 was inoculated in different conditions and the
synthesis of L-amino acid oxidases was evaluated using 2% casamino acids as a substrate. The maximal
production was observed in the supernatant at the stationary phase of growth in medium MNGY
(Supplementary Figure S1).
Next, P. luteoviolacea CPMOR-2 was cultivated until stationary phase in MNGY, and the
supernatant was collected and concentrated using centrifugal filters of a 30 kDa cut-off. These samples
were run under SDS-PAGE in non-denaturing conditions for LAAO activity (see material and methods),
and slices were cut from the gel and evaluated for LAAO and antimicrobial activities. Two protein
bands (fragments 4 and 10) were detected with LAAO activity (Figure 2A). In addition, from a set of
parallel identical lines, those two bands with LAAO activity were excised, and the trypsin was digested,
subjected to HPLC-MS/MS and analyzed against P. luteoviolacea CPMOR-2 genome. It was observed
that various peptides from the upper band matched the protein with GenBank accession number
KZN49687 (with a coverage of 22%). This protein has been named in this work as Pl-LAAO. The
second band with low molecular mass corresponded to PlGoxA (accession WP_063358237) (coverage
of 16%), a LAAO with quinone cofactor specific for glycine, which was recently described [18]. The
upper band showed a strong antimicrobial activity (Figure 2B). Accordingly, we hypothesized that
the upper band may correspond to the LAAO with a broad substrate range previously reported in
P. luteoviolacea CPMOR-2 [16].
Figure 2. Identification of proteins with LAAO and antimicrobial activity in P. luteoviolacea CPMOR-2.
(A) SDS-PAGE of concentrated supernatants of CPMOR-2 strain grown in MNGY medium (See
materials and methods). Arrows point to the protein bands with LAAO activity measured against
casamino acids. LAAO activity for each gel fragment is expressed as relative fluorescence units per
min (RFU/min). (B) Antibiograms against E. coli UM202 of fragment 4 sliced from gel in A showing
antimicrobial activity.
2.2. Sequence Analysis of Pl-LAAO
Pl-LAAO contains 653 amino acids, with a predicted molecular weight of 74 kDa and an isoelectric
point of 4.85 (http://web.expasy.org/compute_pi/). In spite of being detected in the supernatants of the
cultures, Pl-LAAO does not contain any classical or non-classical signal peptide according to the predictor

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web tools SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and SecretomeP 2.0 (http://www.
cbs.dtu.dk/services/SecretomeP). Other web servers, like PSORTb (http://www.psort.org/psortb/)
or CELLO (http://cello.life.nctu.edu.tw/), were not able to provide an accurate prediction of the
subcellular location of Pl-LAAO.
The Pl-LAAO sequence presents a similarity from positions 28–440 to the pfam01593 domain,
which is characteristic of amino oxidases. The sequence analysis also showed in Pl-LAAO some
conserved motifs of the FAD/NAD(P)-binding domain superfamily [20]. For instance, the conserved
dinucleotide binding motif (DBM), with the consensus sequence xhxhGxGxxGxxxhxxh(x)8hxhE(D)
(where x is any residue and h is a hydrophobic residue) is located in the N-terminus (Figure 3A).
In addition, when analyzing the prediction of the secondary structure of this N-terminal region with
JPred4 (http://www.compbio.dundee.ac.uk/jpred/), a structure β1α1β2 is clearly observed, in which
the arrangement of the glycines coincides with the characteristic glycine-rich phosphate-binding
loop of the Rossmann fold [21] (Figure 3B). Shortly after the DBM domain in the Pl-LAAO sequence,
it is possible to recognize the “GG motif” (RxGGRxxS/T), which is a common feature in LAAOs
sequences [22]. Another conserved FAD-binding sequence motif is an eleven amino acid segment,
T(S)xxxxxF(Y)xxGD(E), which has been described in the C-terminal region of proteins from the
glutathione reductase family [20]. The only difference is that this motif in the Pl-LAAO sequence
shows an additional residue between S and F (Figure 3C).
Figure 3. Conserved flavin-binding motifs in Pl-LAAO peptide sequence from P. luteoviolacea CPMOR-2.
(A) The dinucleotide binding motif (DBM) domain (highlighted in grey) and GG-motif (highlighted
in green) in the N-terminal region of the Pl-LAAO sequence. The three conserved Gly in the DBM
domain are in blue, while the two conserved Gly in the GG-motif are in red. The secondary structure
was predicted using the tool “JPred 4” (http://www.compbio.dundee.ac.uk/jpred/). H: alpha helix;
E: beta sheet; -: disordered structure. (
B) First half of the classic Rossmann fold topology. The arrows
designate -strands and rectangles denote
β
α
-helices. ( ) Conserved motif in the C-terminal region
C
(highlighted in red) of proteins from the glutathione reductase family. Conserved residues are in green.
In terms of sequence, as far as we know, no similar protein to Pl-LAAO has been described so far.
For instance, the only FAD-LAAO from a Pseudolteromonas specie reported hitherto, which presents a
homology with L-aspartate oxidases, shares only a 14.3% identity with Pl-LAAO [23]. Achacin, from
the giant African snail Achatina fulica, was found to be the most similar protein to Pl-LAAO that has
been already characterized, with a 20.4% identity [24].
2.3. Recombinant Expression and Partial Biochemical Characterization of Pl-LAAO
To further confirm that Pl-laao encodes the protein with LAAO and the antimicrobial activity
observed in the supernatant of strain CPMOR-2, this gene was cloned into pET15b fused to a poly-His
tag. It was recombinantly expressed in E. coli CD03, which is a strain mutated in a catalase, allowing for
the detection of enzymatic activities releasing hydrogen peroxide in cell extracts [25]. After purification
by a Ni-NTA agarose column and a buffer exchange to remove imidazole, the samples were found to be
yellow, probably indicating the presence of a flavin cofactor. A UV-VIS spectroscopy analysis showed
the characteristic absorption spectrum between 300 and 500 nm that is typical for flavoproteins [26],
with absorption peaks at 370 and 460 nm (Figure 4A). The recombinant purified Pl-LAAO sample was

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subjected to LAAO activity assays with the 20-standard protein
α
-L-amino acids in order to determine
the substrate spectrum. It was observed that Pl-LAAO shows a broad substrate range, with L-Gln and
L-Leu being the preferred substrates. Pl-LAAO also oxidizes L-Glu > L-Met > L-Ile > L-Arg > L-Phe >
L-Tyr > L-Ala > L-Val > L-Lys > L-Trp > L-His > L-Asn > L-Ser > L-Thr and L-Cys (Figure 4B).
Figure 4. (A) UV-VIS spectrum of purified recombinant Pl-LAAO enzyme solution. (B) LAAO activity
spectrum of Pl-LAAO. Values are expressed as percentage of the activity on the best substrate. The
20-protein standard amino acids were assayed at 2 mM, but only the oxidized ones are shown.
A purified sample of the recombinant Pl-LAAO was run in SDS-PAGE. Two bands were observed,
one of them showed the expected molecular mass of the monomer conformation (74 kDa). The second
band with a high molecular mass may correspond to a Pl-LAAO multimeric complex, probably a
homotetramer, according to its apparent molecular mass. The gel was sliced in different fragments for
antimicrobial and enzymatic activity measurements. The results revealed that only the multimeric
form (slice number 1) exhibited the LAAO and antimicrobial activity (Figure 5). The monomeric form
did not present either antimicrobial or LAAO activity. This result indicates that the active conformation
of the enzyme is a multimeric complex, which is in agreement with the size of the active proteins in
the gels of the supernatants of P. luteoviolacea (Figure 2).
To confirm that the antimicrobial activity was due to the hydrogen peroxide generated by the
LAAO activity, distinct experiments were performed. First, it was observed that the antibacterial effect
of Pl-LAAO was inhibited by catalase in the antibiograms against E. coli UM202 in a Luria Bertani (LB)
medium (Figure 6A). In the chemically defined medium M9, no antimicrobial activity was detected
unless amino acids were supplemented. It was observed that the antimicrobial activity correlated with
the hydrogen peroxide production in the oxidation of the amino acid by the LAAO. Thus, L-Gln and
L-Leu, which are the preferred substrates of Pl-LAAO, produced the bigger halos, whereas the Gly
addition did not allowed for the detection of the inhibition halo, as it is not a substrate for the enzyme
(Figure 6B). These results suggest that hydrogen peroxide mainly responsible for the antimicrobial
activity, although it cannot be completely ruled out that some intermediates may potentiate its effect,
similarly to the case of some lysine alpha-oxidases [27].

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Figure 5. Detection in non-denaturing SDS-PAGE of recombinant Pl-LAAO activity. (A) SDS-PAGE of
crude extract and purified Pl-LAAO samples. (1) Crude extract of E. coli CD03 containing pET-15b with
no insert. (2) Crude extract of E. coli CD03 containing pET-15b with Pl-laao as an insert. (3) Purified
Pl-LAAO sample with no treatment. (4) Purified Pl-LAAO sample boiled at 95 ◦C for 5 min. (
B) A
parallel lane similar to lane 3 was sliced to perform antibiograms against E. coli UM202 in a Luria-Bertani
(LB) medium.
Figure 6. Antibiograms of purified recombinant Pl-LAAO against E. coli UM202. (
A
) Antibiograms in
−2
LB. Pl-LAAO disk loaded with 20
20
L of catalase 10 mg mL−1. (
µ
L of purified protein at 7
×
10 mg mL−1. CAT—disk loaded with
µ
B
) Antibiogram in M9 medium in the presence of some amino acids
at 50 mM as a substrate. White disks contained 20
acids 10%.
µ
l of Pl-LAAO at 0.25 mg mL−1. Cas—casamino
2.4. Detection, Distribution, and Phylogenetic Analysis of Proteins Similar to Pl-LAAO
In order to identify similar proteins to Pl-LAAO in the microbial genomes, we did a BLASTp
search against the Integrated Microbial Genomes (IMG) database of the genome sequences as of
14 September 2018. Using the Pl-LAAO peptide sequence as a query and a cut-off limit for the E-value
of 1e−10, we found 57 genes encoding similar proteins to Pl-LAAO. A total of eight genes were not
included in the final analysis. Six of them were repetitive genes with different accession numbers,
and the other two coded for small proteins with less than 25% of the query coverage. This gave us

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a final selection of 49 genes encoding proteins similar to Pl-LAAO, none of them characterized so
far (Supplementary Table S1). All of the detected proteins showed a similarity to the amino oxidase
domain pfam01593 in their sequences, and a size between 526 and 685 amino acids (except one protein
with 334 amino acids). Interestingly, most of the microorganisms synthetizing these proteins were
isolated from seawater samples, which suggests that similar proteins to Pl-LAAO may play a role in
the marine environment (Supplementary Table S1).
From all of the detected genomes, Spirosoma fluviale DSM 29961 is the only microorganism
containing two copies of the genes similar to Pl-laao. Regarding their distribution (Table 1), all of them
were detected in bacterial genomes, which belonged to three distinct phyla i.e., Bacteroidetes (with
18 genes), Nitrospinae (1 gene), and Proteobacteria (30 genes). Within the latter, 28 genes belonged the
Pseudoalteromonas genus, which represents the 20.4% of the total sequenced genomes of this taxon.
If we consider only the Pseudoalteromonas luteoviolacea sequenced genomes, 66.6% of them contained
Pl-laao similar genes (14/21).
Table 1. Occurrence of genes similar to Pl-laao in microbial genomes deposited in the Integrated
Microbial Genomes (IMG) database as of 14 September 2018, according to their phylogenetic
distribution. Phyla are in bold and classes marked with an asterisk. The number between brackets
indicates the total number of sequenced genomes for each taxon at the moment of the analysis.
Phylogenetic
Group 1
Phylogenetic
Group 2
Phylogenetic
Group 3
Taxon
Ungrouped
Proteobacteria (29907)
* Alphaproteobacteria (4218)
* Gammaproteobacteria (19977)
* Betaproteobacteria (3182)
Bacteroidetes (2483)
* Saprospiria (15)
28
0
28
0
0
0
0
0
0
0
0
0
0
0
8
0
0
8
0
0
0
8
1
1
0
0
5
2
3
0
0
1
1
7
1
0
0
1
5
0
0
4
1
0
0
6
* Flavobacteriia (1009)
* Cytophagia (229)
* Unclassified (216)
Nitrospinae (54)
* Nitrospinia (21)
0
28
TOTAL
With the aim of investigating the phylogenetic relationships between the proteins similar to
Pl-LAAO, they were aligned with MUSCLE (MUltiple SEquence Comparison by Log-Expectation),
and then an evolutionary analysis was conducted using the software MEGA6 [28]. Phylogenetic
relationships were inferred using both the neighbor-joining (NJ) and maximum likelihood (ML)
methods. Three different phylogenetic groups, meeting the criterion of being supported by bootstrap
values higher than 70% in both methods, were established (Figure 7).
Group 1 contains 28 proteins, all of them codified by genes from the Pseudoalteromonas genus. The
proteins synthesized by the P. luteoviolacea strains, among them Pl-LAAO, form a well-defined cluster
(Supplementary Figure S2). Interestingly, the analysis of the genome region surrounding the genes
coding for these P. luteoviolacea proteins revealed that all of them showed a similar gene organization
(Figure 8). This organization may suggest that Pl-laao-like genes could form part of an operon, as
they are located together with five other genes similarly orientated with short intergenic regions
between each other. One of the genes in the putative operon encodes an indolepyruvate decarboxylase
related with the Tryptophan metabolism, two encode proteins with a sequence similarity to Spondin_N
pfam06468, and the other two genes code for a response regulator and a histidine kinase, respectively
(Figure 8).

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Phylogenetic Group 2 comprises eight proteins encoded by bacteroidetes from the Cytophagales
order, of which six of them are synthesized by bacteria of the genus Algoriphagus (Figure S3). All
but one presented the conserved COG1231 described in the monoamine oxidases, which is related to
amino acid transport and metabolism. Group 3 represents the most heterogeneous group, consisting of
seven proteins that belong to three different phyla, namely: Bacteroidetes, Proteobacteria, and Nitrospinae
(Figure S4). This may suggest that this group has an ancient evolutionary origin.
With the intention of shedding light on the evolution of Pl-LAAO similar proteins, we performed
a phylogenetic analysis between representative proteins from the three clusters presented above
and the characteristic amino acid oxidases previously reported [5]. Among them, the microbial
proteins for which the encoding gene has been cloned, as well as other representative proteins from
higher organisms are included. The results revealed that the proteins similar to Pl-LAAO constitute a
well-defined cluster and are phylogenetically distant from other LAAOs with antimicrobial activity
described in marine bacteria, like those belonging to the LodA-like group (Figure 9). In fact, proteins
similar to Pl-LAAO are more phylogenetically related to gastropod enzymes than to any other group.
This suggests that Pl-LAAO similar proteins and gastropod LAAOs have a common ancestor, which
has evolved to meet the LAAO and antimicrobial activity in both phylogenetic clusters.
Figure 7. Phylogenetic relationships of Pl-LAAO similar proteins. The tree was created by the
neighbor-joining (NJ) method integrated in the program MEGA6. The sequences were aligned using the
program MUSCLE built in MEGA6. The evolutionary distances were computed using the p-distance
method and are in the units of the number of amino acid differences per site. Numbers at the branches
indicate bootstrap values higher than 70% for both NJ and maximum likelihood (ML) trees.

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Figure 8. General genome region surrounding Pl-laao-like genes in P. luteoviolacea strains. (1) Hypothetical
protein. (2) Tetratricopeptide repeat (TPR)-like response regulator. (3) Indolepyruvate decarboxylase.
(4) and (5) Spondin_N similar proteins. (6) OmpR family response regulator. (7) Signal transduction
histidine kinase. (8) Alcohol dehydrogenase. (9) Multidrug and toxic compounds extrusion (MATE)
family efflux protein. Color code is for cluster of orthologous groups (COG) function category:
Pink—carbohydrate transport and metabolism; green—transcription; gray—signal transduction
mechanisms; light brown—unknown.
Figure 9. Phylogenetic relationships between proteins similar to Pl-LAAO and representative amino
acid oxidases [5]. The tree was created by the NJ method integrated in the program MEGA6. The
sequences were aligned using the program MUSCLE built in MEGA6. The evolutionary distances were
computed using the p-distance method and are in the units of the number of amino acid differences per
site. The numbers at the branches indicate bootstrap values higher than 70% for both of the NJ and ML
trees. Among the Pl-LAAO similar proteins, we selected two proteins representing each phylogenetic
group. For Group 1: Algoriphagus sp. ARW1R1 Ga0206402_101228 (1) and Spirosoma fluviale DSM
29961 Ga0170416_5792 (2). For Group 2: Tenacibaculum ovolyticum DSM 18103 H518DRAFT_00204 (3)
and Lewinella persica DSM 23188 B036DRAFT_03117 (4). For Group 3: Pseudoalteromonas luteoviolacea
CPMOR-2 Pl-LAAO (5) and Pseudoalteromonas luteoviolacea 2ta16 PL2TA16_02145 (6). LAAOs—L-amino
acid oxidases; DAAO—D-amino acid oxidases; LASPOs—L-aspartate oxidases.

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3. Discussion
Several marine microorganisms have been described with an antimicrobial activity, in which
distinct compounds of a different nature are involved. Pseudoalteromonas luteoviolacea is a marine
gammaproteobacteria that had been reported to synthesize antibacterial proteins [16,29]. Here, we
report for the first time the identification, cloning, and heterologous expression of the gene encoding
this antimicrobial protein in P. luteoviolacea CPMOR-2, which has been named Pl-LAAO. Pl-LAAO is a
flavoprotein of 653 amino acids that possesses L-amino acid oxidase activity with a broad substrate
range (Figure 4). The antimicrobial effect of Pl-LAAO was inhibited by catalase, and is directly
proportional to the hydrogen peroxide production, as larger inhibition halos of growth were found
with the preferred substrates (Figure 6). This result suggests that the hydrogen peroxide released in
the amino acid oxidation is responsible for the antimicrobial activity of Pl-LAAO, in agreement with
previous studies [16]. The accumulation of reactive oxygen species like H2O2 may trigger different
forms of cell damage, including lipid peroxidation and DNA strand breakage, which results in bacterial
growth inhibition and cell death. However, it cannot be ruled out that other factors apart from H2O2
could be involved in the antibacterial action of Pl-LAAO. In this sense, it has been described that
certain intermediates may potentiate the antimicrobial effect of some lysine alpha-oxidases [27]. In the
case of the LAAO from Trichoderma harzianum, this enzyme interacts with bacteria, causing membrane
permeability [9].
An interesting feature of Pl-LAAO is that it is still active after SDS-PAGE conditions, which
allowed for the identification of the protein as well as an estimation of its molecular conformation as a
tetramer (Figures 2 and 5). A plausible explanation might be that Pl-LAAO is electronegative at the
pH in phosphate buffer, and according to its theoretical pI value, the protein could not be surrounded
by SDS, thus avoiding the protein unfolding. This resistance to SDS and β-mercaptoethanol has
been described in other flavoproteins with LAAO activity, like the one synthesized by the marine
flavobacterium Aquimarina sp. antisso-27 [17], or by the fungus Rhizoctonia solani, which is indeed
activated by SDS [26].
Previously, various microbial LAAOs with a quinone cofactor had been found with antimicrobial
activity, such as the L-lysine
ε-oxidases from the marine gammaproteobacteria Marinomonas
mediterranea [30], Pseudoalteromonas tunicata D2 [12], Pseudoalteromonas flavipulchra JG1 [13] and
Rheinheimera aquatica GR5 [31]. To the best of our knowledge, this is first report on a FAD-containing
LAAO with antimicrobial activity from a marine microorganism. Additionally, no protein with a
sequence similarity to Pl-LAAO has been cloned or characterized at the molecular level so far. Hence,
a BLASTp was performed against the Integrated Microbial Genomes (IMG) database to detect proteins
similar to Pl-LAAO. A total of 49 genes were detected and, according to their phylogenetic distribution,
the Pl-laao similar genes seem to have no ancient origin as they are not widespread in bacterial genomes,
being detected only in three distinct phyla (Table 1). From the 49 detected proteins similar to Pl-LAAO,
28 were encoded by genes detected in the genomes from the genus Pseudoalteromonas and, specifically,
14 from Pseudoalteromonas luteoviolacea (Table 1). This represents 20.4% and 66.6% of the sequenced
strains for each taxon, respectively. The phylogenetic analysis showed that similar proteins to Pl-LAAO
from the genus Pseudoalteromonas constitute a well-defined cluster of proteins, suggesting that they
have a common evolutionary origin (Figure 7 and Supplementary Figure S2). This fact and their high
abundance in the Pseudoalteromonas genus indicate that this type of proteins may play an important
role in these microorganisms, so they have been subjected to a selective pressure to be conserved
throughout the evolution, particularly in P. luteoviolacea.
Recently, it has been reported that P. luteoviolacea CPMOR-2 synthetizes a LAAO very specific for
Gly, called PlGoxA [18]. This glycine oxidase contains a cysteine tryptophylquinone (CTQ)-cofactor
and belongs to the LodA-like family of quinoproteins [4]. Pl-LAAO and PlGoxA show a low sequence
identity (15.4%) and, curiously, Gly was one of the few substrates not oxidized by Pl-LAAO (Figure 4).
This indicates that both enzymes may perform complementary roles in CPMOR-2. LodA-like proteins
show a L-amino acid oxidase activity, and the hydrogen peroxide produced in the reaction confers

Mar. Drugs 2018, 16, 499
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to them the antimicrobial properties related to microbial biofilm development and dispersal [12].
Similarly to the Pl-laao genes, the occurrence of lodA-like genes in sequenced Pseudoalteromonas and
P. luteoviolacea genomes is also very high (36.5% and 90.5%, respectively). In fact, all of the detected
Pseudoalteromonas genomes containing Pl-laao genes also presented one or various lodA-like genes.
Further studies are necessary in order to understand the relationship between these proteins, which
show important biochemical differences in terms of the cofactor used, but have evolved analogously
to have LAAO and antimicrobial activities.
Regarding the physiological function of Pl-LAAO, several possibilities can be considered based in
the enzymatic reaction catalyzed. Several fungal LAAOs have been proposed to be involved in the
utilization of amino acids as a nitrogen source. Similarly to Pl-LAAO, they have a broad substrate
range allowing the use of several amino acids for growth [32,33]. In addition, the presence of a gene
encoding a putative protein similar to an indolepyruvate decarboxylase related to Trp metabolism, in
the same operon as Pl-laao (Figure 8), suggests the involvement of Pl-LAAO in such a catabolic role as
after the amino acid deamination performed by the LAAO, a keto acid would be generated. On the
other hand, the generation of hydrogen peroxide and the microbial activity associated suggest that
Pl-LAAO and similar proteins could take part in the microbial interaction and competition in microbial
communities, and therefore they may play an ecological role in such interactions. In this regard, the
LAAOs from different gastropods, like the sea hare Aplysia californica and the giant snail Achatina fulica,
have been found to provide protection against invading bacteria [24,34]. In fact, the phylogenetic
analysis revealed that Pl-LAAO and alike proteins are more phylogenetically related to gastropod
LAAOs than to any other kind of enzymes described with amino acid oxidase activity (Figure 9). This
suggests that Pl-LAAO similar proteins and gastropod LAAOs might have a common evolutionary
origin, raising interesting questions about the physiological relevance of these enzymes. Interestingly,
the Pl-laao genes were found not only in the genomes from the Pseudoalteromonas species, but also in
other microorganisms related to host-associated microbial communities from marine organisms, such
as algae, plants, cnidarian, or fishes (Supplementary Table S1). LAAOs with a quinone cofactor from
the marine gammaproteobacteria P. tunicata and M. mediterranea [12] play a role in the development
of microbial biofilms in those surfaces, so it cannot be ruled out that they play a similar role to the
proteins described in this study.
In conclusion, this work shows the identification, cloning, partial characterization, and
evolutionary analysis of Pl-LAAO, the antimicrobial protein described in P. luteoviolacea CPMOR-2. We
believe that our findings provide insights into the biological and ecological significance of antimicrobial
LAAOs in the Pseudoalteromonas genus. The approach and methods described in this investigation
may be used as a guide for the detection of new antimicrobial LAAOs, whose study may be useful for
biotechnological applications.
4. Materials and Methods
4.1. Strains, Culture Media, Plasmids, and Primers
The bacterial strains, plasmids, and primers used in this study are listed in Table 2.
Pseudoalteromonas luteoviolacea CPMOR-2 was isolated from seawater samples [16]. This strain was
◦
usually incubated in a liquid marine media at 25 C and 130 rpm. Different media were used, as
follows: Marine broth 2216 (Difco), complex Marine Medium (MMC) [35], chemically minimal medium
MN [36], MNG (MN plus glucose) [37], and MNGY (MNG plus 0.01% of yeast extract). Escherichia coli
strains were grown in a Luria–Bertani (LB) medium at 37 ◦C and 250 rpm. The LB and M9 chemical
defined medium [38] were used for the antibiograms. When required, the media were supplemented
with the appropriate antibiotic (Sigma-Aldrich, St. Louis, MO, USA).

Mar. Drugs 2018, 16, 499
12 of 16
Table 2. Bacterial strains, plasmids and primers used in this work.
Relevant Genotype and Description,
Strains
Reference or Source
or Sequence
Pseudoalteromonas luteoviolacea CPMOR-2
Escherichia coli CD03
Escherichia coli UM202
Plasmids
Wild-type
[16]
[25]
[39]
BL21(DE3) katE12::Tn10 katG::Tn5, [Cat+/−
]
MP180 katG::Tn10, [Cat+/−
]
pET15b
pET15b
Novagen
pETpl-laao.15
pET15b, Pl-laao
This study
Primers 1
0 0
5 -AAGGAATACATATGACACATTATACTTTTGG-3
5 -CTTCTAACGGATCCTTAAAGTAATCTG-3
AminoORCPMOR2Nde (D)
AminoORCPMOR2Bam (R)
1
0
0
D: direct primer; R: reverse primer; Restriction sites are underlined; [Cat+/−]: decreased catalase activity.
4.2. DNA Manipulations
The DNA was manipulated according to standard protocols [38]. The Pl-laao gene was
amplified from the genomic DNA of the P. luteoviolacea CPMOR-2 genome, using the primers
indicated in Table 2, and the KOD DNA polymerase (Merck, Darmstadt, Germany). The restriction
enzymes were purchased from Fermentas (Thermo-Fischer Scientific, Waltham, MA, USA). The DNA
restriction fragments were eluted from agarose gels by utilizing Qiaquick columns (Qiagen, Venlo,
The Netherlands), and were cloned into a Novagen pET15b plasmid (Merck, Darmstadt, Germany)
using the T4 DNA ligase from Invitrogen. The transformation of E. coli CD03 was carried out by
electroporation [40]. The isolation of plasmid DNA from E. coli was achieved using the Wizard Plus
SV Minipreps DNA Purification System from Promega. The construction was checked by sequencing.
4.3. Expression and Purification of Recombinant Protein
One colony of Escherichia coli strain CD03 containing pET15b with Pl-laao gene fused to a
N-terminal hexahistidine tag was incubated until it reached an OD600~0.6 in an LB medium, with the
addition of 50 µ
g/mL ampicillin at 37 ◦C and 250 rpm. Next, the protein expression was induced
with isopropyl-
β
-D-thiogalactopyranoside (IPTG) 1 mM. Then, the cultures were incubated overnight
◦
at 25 C, before they were harvested by centrifugation at 5000
resuspended in a binding buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4)
and was disrupted by sonication using a Braun Labsonic M sonicator. The homogenate was centrifuged
×
g for 10 min. The cell pellet was
at 13,000
×
g for 2 min, and the pellet was discarded. A cell lysate containing the soluble proteins
was applied to columns with 1.5 mL of Qiagen Ni-NTA resin. The proteins attached to the column
were eluted with an elution buffer (50 mM sodium phosphate, 500 mM NaCl and 500 mM imidazole,
pH 7.4). Amicon® Ultra centrifugal filters 30K (Merck, Darmstadt, Germany) were applied to remove
the imidazole.
4.4. SDS-PAGE
Sodium dodecyl sulfate polyacrylamide (SDS-PAGE) was performed by the method of Laemmli
(1970) [41]. The stacking and separating gels consisted in acrylamide of 3% and 8%, respectively. The
running buffer was 0.3% Tris, 1.44% glycine, and 0.1% SDS, with pH 8.3. The samples were mixed
with a 1/2 volume of loading buffer containing 3 M 2-mercaptoethanol, 0.18 M Tris-HCl, pH 6.8,
15% glycerol, 0.075% bromophenol blue, and 9% SDS. The gels were run at a low voltage (70–90 V).
After electrophoresis, the gels were stained with Coomassie brilliant blue R250. Duplicate lanes with
identical samples were fixed for 2 h in a solution of 10% acetic acid and 20% isopropanol, and were
washed for 2 h in deionized water in order to detect the antibacterial activity of the bands [42]. These
conditions utilized, without boiling the samples with the proteins, which were previously reported
as non-denaturing conditions for the detection of antimicrobial and amino acid oxidase activity [19],

Mar. Drugs 2018, 16, 499
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were non-denaturing conditions for assessing the Pl-LAAO activity as well. The boiling of the sample
resulted in the denaturalization of the protein.
4.5. Activity Assays
To detect the amino acid oxidase activity, a fluorimetric assay for the determination of the H2O2
production was routinely used (Amplex Red hydrogen peroxide/peroxide assay; Invitrogen) [
assay mixture (100 L) contained the substrate (at the specified concentration) in a 50 mM sodium
phosphate buffer with NaCl 0.5 M pH 7.4, 0.05 mM Amplex Red, 0.1 U/mL of peroxidase, and 10 L of
6]. The
µ
µ
sample. Reactions were carried out at 37 ◦C for 15 min in 96-well microplates. Amplex Red oxidation
was followed using an excitation filter of 550 nm and an emission filter of 590 nm in a FLUOstar Optima
(BMG LabTech, Ortenborg, Germany). The activities were normalized according to the milligrams
of protein present in each sample, and were measured by the Bradford assay (Sigma, St. Louis, MO,
USA). The fluorometric assay was also used to determine directly the amino acid oxidase activity in
the fragments sliced from SDS-PAGE, which were previously fixed and washed with deionized water,
as previously described [43].
The antibacterial activity was assayed through antibiograms. A suspension of E. coli UM202 [39
]
in NaCl 0.85% (OD600 = 0.21) was seeded on LB and M9 plates. In some experiments, 20 L of
µ
purified protein, catalase or substrate were loaded into 6 mm disks of Filter Paper Backing (BioRad,
Hercules, CA, USA) and allowed to air dry, before placing them onto the agar plate. To determinate
the antimicrobial activity of the proteins run by SDS-PAGE, the gel was sliced and placed onto the
antibiogram plate after fixing and washing [43]. The antibiograms plates were incubated for 48 h at
25 ◦C.
4.6. UV-VIS Spectrum
The experiments were performed with purified Pl-LAAO in 50 mM phosphate buffer (pH 7.4)
The emission spectra were recorded in a quartz cuvette with a path length of 10 mm using a FLUOstar
OPTIMA fluorescence spectrometer (BMG Labtech, Ortenborg, Germany) at 25 ◦C, and were then
subjected to ultraviolet-visible absorption spectrum analysis. A mixture without Pl-LAAO was used
as a negative control.
4.7. Mass Spectrometry Analysis
The mass spectrometry analysis of protein gel fragments, which consisted of in-gel trypsin
digestion followed by a HPLC-MS/MS analysis, was performed as previously described [19].
4.8. Detection, Alignment, and Phylogenetic Analysis of Pl-LAAO Similar Proteins
In order to identify and analyze the proteins similar to Pl-LAAO (accession KZN49687) in the
microbial genomes, the tools available at the Integrated Microbial Genomes Expert Review (IMG/MER)
were used [44]. BLASTp search at IMG/MER, using an E-value cut of 1e−10 and a minimum identity
percentage of 30%, was performed with the Pl-LAAO sequence as a query (accession KZN49687).
Peptide sequences similar to Pl-LAAO were aligned using MUSCLE (MUltiple Sequence Comparison
by Log-Expectation) [45] and were then incorporated into the program MEGA6 [28] in order to
perform the phylogenetic analysis. The phylogenetic relationships were computed using both the
neighbor-joining (NJ) and maximum likelihood (ML) methods. The distances between the sequences
were computed using the p-distance method and are in the units of the number of amino acid
differences per site. The reliability of each node in the tree constructed was estimated using a
bootstrap analysis with 500 replicates. The proteins analyzed in this study were clustered in different
phylogenetic groups, which met the criterion of being supported by a bootstrap analysis with a higher
than 70% reliability in both of the NJ and ML trees.

Mar. Drugs 2018, 16, 499
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Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/12/499/s1:
Figure S1. LAAO activity in the supernatants of strain CPMOR-2 cultivated in different media at the stationary
phase. The LAAO activity was assayed using casamino acids of 2% as a substrate. Figure S2. Phylogenetic
relationships of Group 1, proteins similar to Pl-LAAO in the genus Pseudoalteromonas. The tree was created by
the NJ method integrated in the program MEGA6. Sequences were aligned using the program MUSCLE built
in MEGA6. The evolutionary distances were computed using the p-distance method and are in the units of the
number of amino acid differences per site. Numbers at the branches indicate bootstrap values higher than 70% for
both NJ and ML trees. Pl-LAAO is indicated in red. Figure S3. Phylogenetic relationships of Group 2 of proteins
similar to Pl-LAAO. The tree was created by the NJ method integrated in the program MEGA6. Sequences were
aligned using the program MUSCLE built in MEGA6. The evolutionary distances were computed using the
p-distance method and are in the units of the number of amino acid differences per site. Numbers at branches
indicate bootstrap values higher than 70% for both the NJ and ML trees. Figure S4. Phylogenetic relationships
of Group 3 of proteins similar to Pl-LAAO. The tree was created by the NJ method integrated in the program
MEGA6. Sequences were aligned using the program MUSCLE built in MEGA6. The evolutionary distances were
computed using the p-distance method and are in the units of the number of amino acid differences per site.
Numbers at branches indicate bootstrap values higher than 70% for both NJ and ML trees. An asterisk indicates
that this branch was not detected, or it had a value lower than 70% in one of trees. Table S1. Similar proteins to
Pl-LAAO, deposited in the Integrated Microbial Genomes (IMG) database as of 14 September 2018.
Author Contributions: Conceptualization, A.S.-A. and J.C.C.-B.; methodology, A.S.-A. and J.C.C.-B.; investigation,
A.A.-V. and J.C.C.-B.; resources, A.S.-A.; writing—original draft preparation, A.A.-V. and J.C.C.-B writing—review
and editing, A.A.-V., A.S.-A. and J.C.C.-B.; funding acquisition, A.S.-A.
Funding: This work has been supported by Ministerio de Ciencia e Innovación, Spain, grant BIO2010-15226,
Co-financed by the European Commission, FEDER funds, and Fundación Séneca, CARM, Spain, grant
11867/PI/09.
Acknowledgments: We thank our lab colleagues for helpful suggestions. We thank Alejandro Torrecillas and the
Molecular Biology Service of the University of Murcia for their support in MS analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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