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Genome Informatics 14: 589–590 (2003)
589
Novel Types of Two-Domain Multi-Copper Oxidases:
Possible Missing Links in the Evolution
Kensuke Nakamura1
Takeshi Kawabata1
[email protected]
[email protected]
Kei
Yura2
Nobuhiro Go1,2
[email protected]
1
2
[email protected]
Graduate School of Information Science, Nara Institute of Science and Technology,
8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan
Quantum Bioinformatics Group, Center for Promotion of Computational Science and
Engineering, Japan Atomic Energy Research Institute, 8-1 Umemidai, Soraku-gun,
Kyoto 619-0215, Japan
Keywords: copper binding protein, cupredoxin, cyanobacteria, phylogeny, rhizobia
1
Introduction
Metal ions bound to proteins exhibit various fundamental functions such as catalysis, electron transfer,
oxigen transfer and signal transmission. Despite its importance, only a little of the bioinformatics
resource has been dedicated to the analysis of the interactions between metal ions and proteins,
so far [1]. Copper is one of the essential metal elements. Multi-copper blue proteins exploits its
distinctive redox property of copper ion. There are three different kinds of multi-copper blue proteins
regarding the number of domains they consist of. Nitrite reductase is a two domain multi-copper blue
protein. Laccase and ascorbate oxidases are three domain multi-copper blue proteins. Ceruloplasmin
is a six domain blue proteins. There have been several discussions on the evolutionary process of
these multi-copper blue proteins [2, 3]. Our phylogenetic investigation on the formation process of
these multi-domain proteins led us to postulate presence of some evolutionary intermediates as shown
in Fig. 1. We here report the search and identification of these hypothetical proteins from genome
sequence.
2
Our postulated phylogenetic pathway for
Method
Results
the multi-copperand
blue proteins
is shown in
Fig. 1. Shaded ovals represent a
cupredoxin domain
and black,
meshed and
Our postulated
phylogenetic
pathempty
dots
indicate
type-1,
type-2 and
way for the multi-copper blue
respectively. Copper
proteins
is residues
shown such
in as
Fig.histidine
1. and
binding
Shaded
ovals
represent
a cuprecistein
are easy
to identify
in the multiple
therefore
we meshed
can predict the
doxinalignment,
domain and
black,
presencedotsorindicate
absencetype-1,
of
each
and empty
copper-binding sites just from the
type-2sequence.
and type-3
copper
We then
lookedatoms,
for sequences
respectively.
Copper
that corespond to binding
our postulated
intermediate
proteins ([A],
residues
such as histidine
and[B],
cis- [C]) in
Domain
Duprication
1'
Type2 site
formation
2
1
Cupredoxin
1"
[X]
Blue copper site loss
from the second domain
2
2"
Loss interdomain
sites
[Y]
1'
Type3 site
formation
Blue copper site
loss from the
second domain
Blue Copper Domains
Type1 blue copper site
2'
Type2 interdomain site
1"
Numbers indicates domain number
1
2"
1"
Nitrite reductase
2
1
2"
1
Domain insertion
3
[C] Interdomain sites loss
2
Blue copper site loss Ascorbate oxidase
1
2"
1'
[A]
Type3 interdomain site
2
2
2'
1"
1'
1'
2'
1
2'
Blue copper site
loss from the
first domain
3
2
1
2'
1"
2"
[B]
Triplication
4
Inter domain
site loss
5
2
1
6
Ceruloplasmin
Figure 1: Proposed evolutionary pathway of blue-copper proteins.
tein are easy to identify in the
multiple alignment, therefore we can predict the presence or absence of each copper-binding sites
just from the sequence. We then looked for sequences that corespond to our postulated intermediate
proteins ([A], [B], [C]) in translated genome sequence database (nr), using BLAST program. Eleven
590
binding sites with the hypothetical proteins. One of them
corresponds to type [A]. Six sequences correspond to [B] and
sequences were identified to possess identical domain orgafour correspond to [C]. Patterns of each sequence is depicted in
nization and
copper
sites
the hypothetical
proFig. 2.
Black binding
bar indicate
the with
mulci-copper
oxidase domain
and
teins. One
of them
type [A].
Six sequences
yellow
box corresponds
indicate signaltopeptide
sequence.
empty dots
indicate
presence
of blue
copper in each
Phylogenetic
correspond
to [B]
and four
correspond
to domain.
[C]. Patterns
of
analysis was carried out using the sequence of newly found
each sequence is depicted in Fig. 2. Black bar indicate the
proteins and conventional multi-copper oxidases. The result
mulci-copper
oxidase
and close
yellowrelationship
box indicate
sigindicates
the domain
relatively
between,
nal peptide sequence. empty dots indicate
of are
blue
e oxidasepresence
and B1~B5
also
be related. Phylogenetic
Weak relationship
between was
nitritecarried
reductase
copper infound
eachtodomain.
analysis
and C2~C4 was also observed. This result is also consistent
out using the sequence of newly found proteins and conventional multi-copper oxidases. The result indicates the relatively close relationship between, ceruloplasmin and B6.
Ascorbate oxidase and B1 B5 are also found to be related.
Weak relationship between nitrite reductase and C2 C4 was
me sequence
database,
novel
also observed. This result is also consistent
with
our postypes
of
two-domain
Cu-oxidases
are
identified.
The
tulated evolutionary relationship.
Nakamura et al.
34
379
A1
Halobacterium strain NRC-1 Q9HQF4
Encoded in a 2-ORF operon
(this protein and plastocyanin-like protein)
rhizobia
449
B1
Q92S43
Sinorhizobium meliloti
Encoded in a 4-ORF operon
(hypothetical, hypothetical, this protein and
putative copper oxidase)
21
450
Agrobacterium tumefaciens
Q8U8U7
Encoded in a 3-ORF operon
(hypothetical, hypothetical and this protein)
31
431
B2
B3
Burkholderia fungorum
30
ZP_00029340
445
B4
Bradyrhizobium japonicum USDA NP_768850
Encoded in a 5-ORF operon
(hypothetical, copper tolerance protein, this protein,
copper tolerance protein and hypothetical)
477
28
B5
Magnetospirillum magnetotacticum ZP_00052601
121
onspicuous combination of
M. magnetotacticum
ZP_00049708
tri-nuclear copper binding
32
348
B6
residues, that is common in multi-copper oxidases, such as
Streptomyces griseus
Q93HV5
3 Discussion
367
ceruloplasmin, laccase, and ascorbate oxidase. Therefore
C1
in the characteristics common
Nitrosomonas europaea Neur_1 ZP_00002680
From thewith
analysis
of genomecommon
sequenceancestoral
database, form
novel types
343
a plausible
of the
C2
of two-domain
Cu-oxidases
are
identified.
The
two-domain
NP_711736
Leptospira
interrogans
multi-copper oxidases. Presence of these multi-copper blue
Encoded in a 3-ORF operon
proteins have conspicuous combination
of blue-copper
and
our postulated
evolutionary
(lipoprotein, hypothetical and this protein)
pathways
(Fig. 1).copper
Function
of those
newly that
identified
cyanobacteria
inter-domain
tri-nuclear
binding
residues,
is
331
C3
is hard to
predict. such
However,
considering the
common proteins
in multi-copper
oxidases,
as ceruloplasmin,
Trichodesmium erythraeum IMS101 ZP_00073469
similarity of copper binding sites, we infer the function of
33
338
laccase, and
ascorbate oxidase. Therefore they are considC4
newly found proteins are similar to that of laccase, ascorbate
NP_487982
Nostoc sp. PCC 7120
ered to retain
characteristics
common
a plausioxidasethe
or ceruloplasmin,
rather
than that with
of nitrite
reductase. Figure 2:
Newly found two-domain
ble common ancestoral form of the multi-copper oxidases.
multi-copper oxidases.
Presence of these multi-copper blue proteins strongly supports our postulated evolutionary pathways (Fig. 1). Function of those newly identified proteins is
hard to predict. However, considering the similarity of copper binding sites, we infer the function of
newly found proteins are similar to that of laccase, ascorbate oxidase or ceruloplasmin, rather than
that of nitrite reductase. All eleven sequences are found in bacteria. Most of those organisms such as
rhizobia and halobacteria are known to be capable of nitrogen fixation. Therefore, it is plausible to
consider that function of these proteins are related to the nitrogen fixation.
References
[1] Bertini, I. and Rosato, A., Bioinorganic chemistry in the postgenomic era, Proc. Natl. Acad. Sci.
USA, 100:3601–3604, 2003.
[2] Murphy, M.E.P., Lindley, P.F., and Adman, E.T., Structural comparison of cupredoxin domains:
domain recycling to construct proteins with novel functions, Protein Science, 6:761–770, 1997.
[3] Ryden, L.G. and Hunt, L.T., Evolution of protein complexity: the blue copper-conteining oxidases
and related proteins, J. Mol. Evol., 36:41–66, 1993.