Amylase 2017; 1: 1–11
Research Article
Open Access
Vincent Valk, Rachel M. van der Kaaij, Lubbert Dijkhuizen*
The evolutionary origin and possible functional
roles of FNIII domains in two Microbacterium
aurum B8.A granular starch degrading enzymes,
and in other carbohydrate acting enzymes
DOI 10.1515/amylase-2017-0001
Received October 19, 2016; accepted December 17, 2016
Abstract: Fibronectin type III (FNIII) domains were first
identified in the eukaryotic plasma protein fibronectin,
where they act as structural spacers or enable proteinprotein interactions. Recently we characterized two large
and multi-domain amylases in Microbacterium aurum
B8.A that both carry multiple FNIII and carbohydrate
binding modules (CBMs). The role of (multiple) FNIII
domains in such carbohydrate acting enzymes is
currently unclear. Four hypothetical functions are
considered here: a substrate surface disruption domain,
a carbohydrate binding module, as a stable linker, or
enabling protein-protein interactions. We performed a
phylogenetic analysis of all FNIII domains identified in
proteins listed in the CAZy database. These data clearly
show that the FNIII domains in eukaryotic and archaeal
CAZy proteins are of bacterial origin and also provides
examples of interkingdom gene transfer from Bacteria
to Archaea and Eucarya. FNIII domains occur in a
wide variety of CAZy enzymes acting on many different
substrates, suggesting that they have a non-specific role
in these proteins. While CBM domains are mostly found
at protein termini, FNIII domains are commonly located
between other protein domains. FNIII domains in
carbohydrate acting enzymes thus may function mainly
as stable linkers to allow optimal positioning and/or
*Corresponding author: Lubbert Dijkhuizen, Microbial Physiology,
Groningen Biomolecular Sciences and Biotechnology Institute
(GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen,
The Netherlands; E-mail: [email protected]
Vincent Valk, Rachel M. van der Kaaij, Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
University of Groningen, Nijenborgh 7, 9747 AG Groningen, The
Netherlands
Vincent Valk, Top Institute of Food and Nutrition (TIFN), Niewe Kanaal 9A, 6709 PA Wageningen, The Netherlands
flexibility of the catalytic domain and other domains,
such as CBM.
Keywords: Microbacterium aurum; FNIII domains; protein
evolution; carbohydrate acting enzymes; phylogeny; CBM
Abbreviations: AA, auxiliary activities; CAZy,
Carbohydrate-Active
enZymes
database;
CBM,
carbohydrate binding module; CDD, Conserved Domain
Database; CE, carbohydrate esterases; FNIII, fibronectin
type III; GH, glycoside hydrolases; GT, glycosyl
transferases; PDB, Protein Data Bank; PL, polysaccharide
lyases.
1 Introduction
The Gram-positive bacterium Microbacterium aurum B8.A
was isolated from the sludge of a potato starch-processing
factory on the basis of its ability to use granular starch as
carbon- and energy source for growth [1]. Its extracellular
amylolytic enzymes were able to degrade starch granules,
initially by introducing pores. In biochemical studies we
have characterized MaAmyA and MaAmyB, two large
and multi-domain amylase enzymes. MaAmyA (148 kDa)
carries two carbohydrate binding modules (CBM25),
four fibronectin type III (FNIII) domains and one CBM74
(Fig. 1) [2,3]. Characterization of MaAmyA mutant proteins
with C-terminal deletions of various lengths showed that
the CBM25 domains are essential for the ability of this
enzyme to degrade raw starch. Deletion of the C-terminal
CBM74 in MaAmyA resulted in a three-fold reduction in
granular starch pore size [2,3]. Deletion of CBM25 and
CBM74 negatively affected degradation of raw starch,
but not that of soluble starch [2]. In MaAmyA a total of
three FNIII domains are present between the two CBM25
domains and the CBM74 domain (Fig. 1). It has remained
© 2017 Vincent Valk et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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2 V. Valk, et al.
unclear what the precise function is of these three FNIII
domains. MaAmyB (135 kDa) is the first characterized
member of a new glycoside hydrolase family 13 subfamily
(GH13_42), which has a strongly conserved general
domain organization featuring one to four FNIII domains
between the two CBM25 domains and the catalytic domain
(with an aberrant C-region) (Fig. 1) [4]. In both enzymes
the precise function of these four FNIII domains has
remained unclear; therefore we decided to study this in
a more detail, focusing on carbohydrate acting enzymes
in general.
The FNIII domain is an evolutionarily conserved
protein domain of approximately 100 amino acids,
with a β-sandwich structure. It is one of the three
types of internal repeats (FNI-FNII-FNIII), originally
identified in the eukaryotic plasma protein fibronectin.
FNIII domains in eukaryotic proteins usually act as
structural spacers, to arrange other domains in space,
as in fibronectin itself. FNIII domains also may have
functional roles in the formation of protein-protein
interactions [5]. FNIII domains are widely found in
extracellular eukaryotic proteins, especially in animals,
but also occur in plant and yeast proteins. Whereas
other fibronectin repeats (FNI and FNII domains)
only occur in proteins of Eucarya, FNIII domains also
are found in Bacteria, Archaea and viruses. The first
FNIII domains in prokaryotes were reported in 1990
[6]. Due to the low number of recognized bacterial
FNIII domains at that time, and phylogenetic analysis
placing them separate from the eukaryotic domains,
it was suggested that prokaryotes had acquired FNIII
domains from eukaryotes [7]. All initially identified
prokaryotic FNIII domains were associated with
carbohydrate acting enzymes, but more recently these
domains have also been identified in fibronectin
binding proteins and multiple other prokaryotic protein
types [8-10]. Our database searches showed that about
33% of all FNIII containing proteins included in SMART
database (15125) [10] are from bacteria. Approximately
19% of all bacterial FNIII domains occur in proteins
that are directly related to carbohydrates, such as
carbohydrate acting enzymes or proteins that contain
CBMs [10]. In this work we focus on FNIII domains that
are part of carbohydrate acting enzymes classified in
the Carbohydrate-Active enZymes (CAZy) database [11].
Microbial carbohydrate acting enzymes are able to
degrade complex carbohydrates, making them available
as a carbon and/or energy source for growth. Many are
listed in the CAZy database, which shows five different
enzyme classes based on their activities and mode of
action: glycoside hydrolases (GH), glycosyl transferases
(GT), polysaccharide lyases (PL), carbohydrate esterases
(CE) and auxiliary activities (AA) [11]. These classes have
been separated into families and some (larger) families
have been further divided into subfamilies, based on
the primary structure of their protein members [11,12].
Approximately 10% of these enzymes include one or more
CBMs [3,13] (this work).
The function of FNIII domains in carbohydrate
acting enzymes is currently unclear. So far no reciprocal
protein-protein interactions have been reported to exist
between bacterial FNIII domains, although an interaction
has been suggested to exist between fibronectin binding
proteins FNIII domains and eukaryotic fibronectin
[9]. Characterization of different carbohydrate acting
enzymes resulted in three suggestions for the role of their
FNIII domains: as stable linker [14], carbohydrate surface
disruption domain [15] and CBM [16].
In this work we traced all FNIII domains present in all
carbohydrate active enzymes listed in the CAZy database
of February 2016, most of which (98%) were from bacterial
origin. We used both bioinformatics approaches as well
as the available literature to analyse the possible roles of
FNIII domains in carbohydrate acting enzymes.
MaAmyA
1,417
MaAmyB
GH13_42
1,278
993
Figure 1. Domain organization of MaAmyA [2], MaAmyB [3] and the general domain organization of GH13_42 members. Numbers refer to
the number of amino acids in the protein. The length of GH13_42 varies for each member, as a reference the length of AmlC from S. lividans
(GenBank: CAB06816.1) is shown. Domains are indicated as follows: white, signal sequence; red, AB-regions of the catalytic domain;
pale yellow, C-region; blue, FNIII domain; green, CBM25 domain; pink, CBM74, refers to a novel CBM domain [3]; orange yellow, aberrant
C-region.
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2 Materials and methods
2.1 Extracting FNIII domain sequences from
all CAZy entries
We extracted the (linked) GenBank accession number for
each CAZy entry in the 01-28-2016 release [11]. When no
linked GenBank accession number was found, the linked
Protein Data Bank (PDB) entry was used instead. If neither
was present, the best matching sequence of the non-linked
entries was used. In case no database links were listed, we
attempted to trace the sequence in the databases using the
name and strain information. Obsolete sequences were
updated to their newer version as suggested by GenBank, if
available [17]. The accession numbers were used to extract
the corresponding protein FASTA sequences from GenBank.
All FASTA sequences were separated in files of 25,000
sequences and then submitted to batch CD-search, using
the live search option and Conserved Domain Database
(CDD) database [18]. The concise output was used and all
lines containing “specific” in the “Hit type field” and “FN3”
(FNIII domains are designated as FN3 in CDD) in the “short
name field” were extracted. The lines contained the start
and end amino acids for each FNIII domain. Microsoft
Excel 2010 was used to extract the sequence of each FNIII
domain from the full FASTA file. Since some sequences
contained more than one FNIII domain, sequences of FNIII
domains were named as follows: “accession number of
full sequence”-“start aa number of the FNIII domain” The
output was formatted as a FASTA file.
2.2 Extracting FASTA sequences for all CBMs
listed in CAZy
All enzymes containing one or more CBMs according to the
CAZy annotation in the 01-28-2016 release were extracted
as FASTA sequences, as described above. All FASTA
sequences were separated in files of 2,500 sequences
and submitted to dbCAN [19]. The output contained the
domain organization of the full-length proteins with
the positions of the CBMs up to CBM67 as annotated by
dbCAN. CBM families 68 and higher are currently not
included in dbCAN and are therefore not included in the
analysis, except for CBM74, which we annotated manually
based on our previous work [3].
2.3 Bioinformatics tools
All BLAST searches were performed with NCBI BLASTP
[20] using standard settings. Conserved domains were
detected using both the NCBI conserved domain finder
FNIII domains in carbohydrate acting enzymes
3
[18] (with forced live search, without low-complexity filter,
using the CDD), and dbCAN [19] with standard settings.
Alignments were made with Mega6.0 [21] using its build-in
muscle alignment with standard settings. Alignments
were visualized with Jalview 2.8.1 [22]. Phylogenetic trees
were made with Mega6.0 using maximum likelihood
method with gaps/missing data treatment set on partial
deletion instead of full deletion. Trees were visualized
with Interactive Tree Of Life v2 [23]. Information about
enzyme activity (EC number) was obtained from the CAZy
database [11]. Domain organization shown in the tree is
based on combined data of dbCAN and CDD. Enzyme class
information was obtained from CAZy. Secondary structure
prediction was done using the Phyre2 server with standard
settings [24].
2.4 Alignment and phylogenetic tree
An initial alignment and tree using all 3,219 FNIII domain
sequences extracted from proteins in the CAZy database
was constructed. Due to the high number of sequences
the alignment was not manually tuned but directly used to
produce a phylogenetic tree. This large tree was used to select
a smaller number of sequences including all eukaryotic and
all archaeal FNIII domains and a diverse selection (based
on the initial tree) of bacterial FNIII domains as present
in CAZy proteins. These selected sequences were used
for an alignment, which was manually tuned and used
to produce the phylogenetic tree shown (Fig. 2). For each
selected protein, all FNIII domains present were included
in the tree. A total of 107 proteins with 158 FNIII domains
were selected and used for the alignment and phylogenetic
tree construction.
Two additional phylogenetic trees were constructed
based on a random selection of FNIII domains found in
an equal number of proteins from each of the 4 kingdoms
(Archaea, Bacteria, Eucarya, viruses), obtained from
the SMART database [10]: one tree based on 100 FNIII
domains (Fig. S1), and one based on 1,000 domains (data
not shown).
2.5 Determination of carbohydrate related
FNIII domains
The FASTA files of all bacterial FNIII containing protein
sequences listed in the SMART database were extracted.
All 15,125 bacterial protein sequences were analysed (in
batches of maximal 3,000 sequences) with dbCAN. Results
were screened for carbohydrate related catalytic domain
hits. Proteins were considered carbohydrate related when
such a catalytic domain was identified in the sequence.
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4 V. Valk, et al.
Legend:
Shapes:
Complete domain
Incomplete domain
Domain incomplete N-terminus
Domain incomplete C-terminus
Position in tree based on aa sequence of this domain
Domain colors:
GH13 amylase AB region
GH13 amylase C region
GH catalytic domain
AA catalytic domain
CE catalytic domain
GT catalytic domain
FNIII
FNIII-Like2
CBM25
CBM74
Other CBM
Other domain
Legend:
C-terminus
Domain
organization
Domain of which the
sequence was used to
determine the position
in the tree
N-terminus
Outer ring
Center ring
Legend.1-111
Start aa no. of sequence used to
determine the position in the tree
Name (accession no.)
Inner ring
branch line
Inner ring colors (indicating genera/species
of the bacteria containing the protein)
0.1
Amycolatopsis
Bacillus
Cellulomonas
Corallococcus
Halorhabdus
Listeria
Microbacterium aurum B8.A
Paenibacillus
Ruminiclostridium
Streptomyces
Yersinia
Other species (present twice in this tree)
Other species (present once in this tree)
Center ring & branch line colors
(indicating a selection of clusters)
Outer ring colors
(indicating kingdom)
Eukaryotic cluster
Archaea
Archaeal cluster
Bacteria
C. thermocellum cluster
Eukarya
Microbacterium aurum cluster
Unclassified
1st FNIII of MaAmyA cluster
Cluster rich in interkingdom horizontal gene transfer
Figure 2. Phylogenetic tree with all eukaryotic, archaeal and (a random selection of) bacterial FNIII containing proteins in CAZy. MaAmyA,
MaAmyB and Clostridium thermocellum CbhA (GenBank: CAA56918.1) were also included. Phylogeny was based on the FNIII sequences as
defined by CDD. All FNIII domain sequences were obtained as described in the Methods section, except for the FNIII domains from CbhA,
which were extracted manually based on the published information [16]. Domain organization is based on combined dbCAN and CDD data.
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FNIII domains in carbohydrate acting enzymes
3 Results and discussion
5
identified were found in proteins that belong to the GH
class (64.2%). This also reflects the fact that nearly half
(48.5%) of all CAZy proteins belong to the GH class.
When we look at the number of FNIII domain containing
proteins in each enzyme class separately, the AA class has
the most FNIII containing proteins (2.3%). Interestingly,
the number of FNIII domains in CBM containing proteins
is even larger: 3.5% of all CBM containing proteins also
have one or more FNIII domains, and 71.7% of all FNIII
containing proteins also contain one or more CBMs (Table
1). This last number is even higher (76.0%) when dbCAN is
used instead of CAZy to annotate the CBM domains. This
clearly points to a link between the presence of FNIII and
CBM domains in proteins.
FNIII domains are conserved protein domains of about
60-120 amino acid residues that have an all β-sheet
secondary structure. In this work, FNIII domains are
defined as domains indicated by CD-search as a “Specific”
hit with the short name “FN3”. All domains matching
these criteria had one of the following defined domain
ID’s: cd00063 [18], pfam00041 [25] or smart00060 [10].
3.1 FNIII domains in carbohydrate acting
enzymes listed in CAZy
In total, we retrieved 575,089 unique FASTA protein
sequences from the CAZy database. These were submitted
to a CD-search, which identified 3,219 FNIII domains,
present in 2,486 CAZy proteins. Less than 0.5% of all
CAZy proteins thus contain one or more FNIII domains,
as defined by the criteria used in this study. Of these 2,486
proteins, 76% contained one FNIII domain, 21% contained
two FNIII domains and 3% contained three or more FNIII
domains, up to a maximum of seven FNIII domains in
proteins CBL14028.1, CBL07763.1 and CBL13779.1. The
3,219 FNIII domains identified in CAZy proteins in this
study is in sharp contrast with the 24 FNIII domains that
thus far explicitly have been reported in characterized
carbohydrate acting enzymes in the literature (see
below). Clearly, little is known about the abundance and
functionality of FNIII domains in CAZy proteins.
FNIII domains were identified in proteins belonging
to all five CAZy enzyme classes (Table 1). This is in contrast
to the specific CBM families, which are usually linked to
one or two different enzyme classes, but so far never to
all five [11]. A relatively large number of the FNIII domains
3.2 Phylogenetic analysis
A phylogenetic tree with all eukaryotic, archaeal and
(a random selection of) bacterial FNIII containing
carbohydrate acting enzymes (with phylogeny based
on the FNIII sequences as annotated by CDD) listed in
CAZy was made (Fig. 2). Two FNIII-like domains from
Clostridium thermocellum were also included (discussed
in the next paragraph). The tree shows six clusters. Three
clusters contain mainly bacterial FNIII domains, another
cluster is of a mixed type with several examples of interkingdom gene transfer (indicated in grey in Figure 2) The
two remaining clusters are clearly distant from the others.
One contains most of the FNIII domains in eukaryotic
CAZy proteins (indicated in green in Figure 2), while the
other contains most of the FNIII domains obtained from
archaeal CAZy proteins (indicated in blue in Figure 2).
There is no clear FNIII clustering evident at the bacterial
genus/species levels. For example, FNIII domains from
Table 1. Distribution of FNIII domains over proteins in the different CAZy enzyme classes.a
Associated
modulesc
CAZy enzyme classesb
AA
CE
GH
GT
PL
CBM
2.0%
5.5%
48.5%
39.4%
1.3%
8.7%
Percentage of proteins per class containing (one or more) FNIII domain(s)
2.3 %
2.3%
0.6%
0.1%
0.7%
3.5%
Number of proteins per class containing (one or more) FNIII domain(s)
264
720
1,596
280
54
1,783
Percentage of all FNIII containing proteins belonging to the class
10.6 %
29.0%
64.2%
11.3%
2.2%
71.7%
Percentages of total CAZy entries per classd
d
d
e
Some proteins in this table belong to multiple CAZy enzyme classes, therefore the percentages shown do not add up to 100%.
GH, Glycoside Hydrolases; GT, Glycosyl Transferases; PL, Polysaccharide Lyases; CE, Carbohydrate Esterases; AA, Auxiliary Activities.
c
The last column indicates the distribution of FNIII domains over CAZy entries with one or more CBM (based on the 01-28-2016 release of
CAZy); as defined by CAZy (http://www.cazy.org/; [11]).
d
Total number of proteins in each class: AA:11731, CE:31583, GH:279001, GT:226733, PL: 7245, CBM:50301.
e
Total number of FNIII domain containing proteins: 2486.
a
b
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6 V. Valk, et al.
similar enzymes obtained from different Streptomyces
species are scattered throughout the tree. This again
suggests that these FNIII domains have been obtained
through horizontal gene transfer. The tree suggests that
the FNIII domains in CAZy proteins originated from
bacteria, since the eukaryotic cluster (indicated in green in
Figure 2) and archaeal cluster (indicated in blue in Figure
2) are further from the origin of the tree. This contrasts to
literature, where it has been stated that prokaryotic FNIII
domains originate from Eucarya [7,26]. To analyse whether
the subset of FNIII in CAZy enzymes is representative
for all FNIII domains, two additional phylogenetic trees
were produced including 100 (Fig. S1) or 1,000 (data not
shown) FNIII domains randomly picked from SMART [10],
but containing an equal number of domains from each
of the four kingdoms. Neither of these two additional
phyloogenetic trees produced showed clear clustering of
FNIII domains according to kingdoms, or a clear origin of
the tree (Fig. S1). Clearly, these trees also do not support
the hypothesis that bacterial FNIII domains were originally
obtained from eukaryotes. This conclusion was reached
in 1992 based on phylogenetic analysis of the 13 bacterial
FNIII domains known at that time, all from carbohydrate
acting enzymes, when compared to 26 eukaryotic FNIII
domains [7]. This conclusion was restated in 2002, when
the first bacterial FNIII domain tertiary structure was
solved, and based on the phylogenetic analysis of 20 of
the 135 bacterial FNIII domains, which were at that time
(July 2001) listed in SMART [10]. This appears to be the last
time that the subject was studied using a bioinformatics
analysis, but subsequently it has been referred to in many
other FNIII domain studies and reviews (for example [2731]). The phylogenetic trees presented in this study (Fig.
2, Fig. S1) do not support the conclusion that Eucarya are
at the root for FNIII domains. Since about 33% of all FNIII
domains currently listed in SMART is from prokaryotes,
also the statement that there are many more eukaryotic
FNIII domains than prokaryotic ones has become
outdated [7,26]. We conclude that on basis of all available
information there is no evidence that bacterial FNIII
domains were originally obtained from eukaryotes.
3.3 FNIII domains as cellulose surface
modifiers
Two FNIII-like domains from Clostridium thermocellum
cellobiohydrolase CbhA (GenBank CAA56918.1) were
shown to act as cellulose surface modifiers [15], making the
smooth and even surface of the cellulose substrate rough
and uneven. These two domains were not among the 3,219
FNIII domains identified in this study, because they were
not identified as such by CD-search [19]. Our additional
Pfam analysis [25] also did not identify them as FNIII
domains. Therefore, these CbhA domains were extracted
manually from the protein sequence, using the published
information [15]. Their sequences were used for alignment
together with all other bacterial FNIII sequences obtained
from characterized carbohydrate acting proteins (Fig. 3).
The two FNIII-like domains extracted from CbhA
only show 7-14% identity and 15-30% similarity to the
24 characterized FNIII domains from the bacterial
carbohydrate acting enzymes included in the alignment
(Fig. 3), while identity among the 24 true FNIII domains is
17-80% (30-84% similarity). In a phylogenetic analysis, the
two CbhA domains do not cluster with other FNIII domains
(Fig. 2). Four residues that are known to be conserved in
FNIII domains [32] are also fully conserved in the FNIII
domains included in the alignment (Fig. 3; highlighted
in blue, pink, yellow and green), while only the leucine
residue is conserved in the two domains from ChbA. We
conclude that these two domains of ChbA, in fact, are not
FNIII domains at all according to the current definitions.
This also is reflected in their listing in CDD, where they
are currently indicated as “FNIII-like2 domains”. Since
the suggestion that FNIII domains function as surface
disruption domains was based only on the experimental
results obtained with these two domains in ChbA, this
hypothesis is now no longer supported by experimental
data.
3.4 Location of FNIII domain in the protein
Since domains acting as stable linkers between other
protein domains are not expected to be found at the
terminus of a protein, the position of FNIII domains
within the 2,486 FNIII containing enzymes listed in CAZy
was studied. For comparison, the position of CBMs in
CAZy proteins (based on annotation of CBM domains with
dbCAN) was included. We defined domains as terminal
domains when there are less than 100 amino acid residues
between the predicted domain sequence and the protein
N- or C-terminus. Also, it was not allowed for the remaining
amino acids to contain a protein domain as identified by
either CD-search or dbCAN.
In total, there are only 332 proteins that have a FNIII
domain either at the N- or at the C-terminus including five
proteins with both an N- and C-terminal FNIII domain.
This is 14% of all FNIII containing proteins. In comparison,
82% of all CBM containing proteins have a CBM located
at the protein terminus (Fig. 4). Only in a small number
of CBM families (13 out of 66 families), the majority of
the CBM members is not located at one of the protein
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FNIII domains in carbohydrate acting enzymes
10
ACR15165.1-927
ACR15165.1-1166
AAA19800.1-931
AAA19800.1-1169
ABY95795.1-928
ABY95795.1-1158
EFB38194.1-652
ACY75514.1-359
ADR71224.1-491
ADR71224.1-570
AAA81528.1-465
AAA81528.1-560
AAU21943.2-460
BAA76623.1-338
AEN08307.1-298
AEN08307.1-391
MaAmyA-530
MaAmyA-833
MaAmyA-925
MaAmyA-1017
MaAMyB-264
MaAMyB-356
MaAMyB-448
MaAMyB-540
First FNIII cellulase
Second FNIII cellulase
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Y
-
L
-
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DE Q
- - -
Y
-
T
-
- - - - - - - - - - - - - - - - - - - - - - - - DS
V I
E
V
E
T
K
P Q A P S N V V V T - - S G NG K V D L
- - - - - - - - - - - -T E S S RVS L
P Q V P S N V V A T - - S G NG K V D L
- - - - - - - - - - - -VE S S RVS L
P Q P I T D L K A V - - S G NG Q V D L
P P T A L G L QQ PG - I E S S R V T L
P AF P TG L S AV L DS S G NT ANL
PS APTGL RVTG - T T T S S I S L
- - - - - - - - - - - - - - - - - - -V
PS APTGL RVTG - T T T S T VAL
P S V PG NAR S TG - V T ANS V T L
P T AP T NL AS T A -QT T S S I T L
- S AP V NV R V TG - K T AT S V S L
P NAP T AL T V AE - L G AT S L K L
P T AP SG L AF T E - P ATGQV K L
P T A P S G L A F T E - P A AG Q I K L
P T V P S N V K A T - - A NG T T I T V
PAVPSGL KAT - - VSGT S VAL
PAVPSGL KAT - - VSGT S VAL
PAVPSGL KAT - - VSGT S VAL
PAVPSGL KAT - - VSGT S VAL
PAVPSGL KAT - - VSGT S VAL
PAVPSGL KAT - - VSGT S VAL
P A V P T G V K T V - - V S GQQ V S V
DK V T I DS P V AG E R F E AG K D I
P T V K L T AP K S NV V AYG NE F L
40
EGG LY E K I AS
ACR15165.1-927 39
ACR15165.1-1166
DACR15165.1-1166
YE I
30
DG G T F N K I A T
T GAAA19800.1-931
YAAA19800.1-931
N I S S V39
EGG LY E K I AS
AAA19800.1-1169
NAAA19800.1-1169
Y E I - Y R - - S30
DG G T F N K I A T
V SABY95795.1-928
YABY95795.1-928
N I - Y R S T V39
KGGL Y E K I AS
ABY95795.1-1158
GABY95795.1-1158
Y E I - Y K S L S44
E TG PF VK I AT
N SEFB38194.1-652
YEFB38194.1-652
N V - K R S T K39
SGGP Y T T I AT
YACY75514.1-359
R V - F R - - -39
GACY75514.1-359
- DG - - T Q V A E
YADR71224.1-491
T V - L V - - -21
- DG - - A A V G T
T GADR71224.1-491
- DG - - T Q V A E
T GADR71224.1-570
YADR71224.1-570
R V - F R - - -39
T GAAA81528.1-465
YAAA81528.1-465
NV - - -38
- -G - - ANL AT
T GAAA81528.1-560
YAAA81528.1-560
DV - - -38
- -G - - T AL AT
EAAU21943.2-460
YAAU21943.2-460
VVS
- - - 38
- - - - - NR S I S
A SBAA76623.1-338
YBAA76623.1-338
T V - - -39
- NG - - N P I G Q
YAEN08307.1-298
D I
A - - -39
- G G - - T L L AG
T GAEN08307.1-298
YAEN08307.1-391
DV
A - - -39
- DN - - V L R K S
T GAEN08307.1-391
MaAmyA-530
MaAmyA-530
T G G43
TGG - - S V T L T
SG YQ
I MaAmyA-833
T K Y RMaAmyA-833
VA
- - -37
DAG V V K Y V T V
MaAmyA-925
T K Y RMaAmyA-925
VA
- - -37
DAG V V K Y V T V
MaAmyA-1017
T K YMaAmyA-1017
RVA
- - -37
DAG V V K Y V T V
T K Y MaAMyB-264
RMaAMyB-264
VA
- - -37
DAG V V K Y V T V
T K Y MaAMyB-356
RMaAMyB-356
VA
- - -37
DAG V V K Y V T V
T K Y MaAMyB-448
RMaAMyB-448
VA
- - -37
DAG V V K Y V T V
T S Y MaAMyB-540
TMaAMyB-540
I S
- - T38
G SGAAT T V AS
First
K V FNIII
EFNIII
F -cellulase
Y N - - -51
-GD- - T L I S S
SFirst
cellulase
Second
I S R FNIII
V D -cellulase
- - V47
DG - - - E V I G S
20
50
NV T E V S NE V
NV TG V S NE V
NV TQ V ADT V
N I T S VAA - T S T - I S A - S VTG T VTG VKE - T AG - V AG DV
V AG DV
S T S - T SG - T SG - T SG - T SG - T SG - T SG - T T - - DT T AP
DR E AP
60
T T F E D A N V T NG L K Y V Y
Y N Y I D T S V I NG V T Y N Y
T T F E D T N V T NG L K Y V Y
Y N Y V D T S V I NG T T Y S Y
I T Y I D T D V T NG L K Y V Y
Y N Y V D T D V V NG K V Y Y Y
T NY T DTG V ATG T K Y Y Y
T S F T DT G L T AG T A H V Y
T S F T V T G L E AG T S Y A V
T S F T DT G L A AG T A H V Y
T T A T I S G L T AG T S Y T F
T T AT I S G L AADT S Y T F
T S AE I GG L K PG T AY S F
L S L T DS G L T P ATQ Y S Y
T T Y T DT R R ADE T - V T Y
T T Y T DT Q - P AG T T V S Y
TQ L I DS G L AE NT T Y S Y
T T L T V S D L A AG T A Y WF
T T L T V S G L S AQ T A Y WF
T T L T V S G L S AQ T A Y WF
T T L T V S D L A AG T A Y WF
T T L T V S G L S AQ T A Y WF
T T L T V S G L S AQ T A Y WF
AAK V I TG L AAS T T Y NF
Y T AK I TG AAVG AY NL K
Y E Y E WK A V E G N H E I S V
7
30
S WS Q S D - - - - G A T G Y N I - Y
T WN P S T D N V - G I Y D Y E I - Y
S WS Q S D - - - - G A T G Y N I - Y
T WS P S T D N V - G I Y N Y E I - Y
S WS A V D - - - - R A V S Y N I - Y
NWS L S T D N V - A I Y G Y E I - Y
T WN A A P - - - - G A N S Y N V - K
AWN A S T D D V - G V AG Y R V - F
S WT A S T D D R - G V T G Y T V - L
AWN A S T D D T - G V T G Y R V - F
AWN A S T D N V - G V T G Y N V - Y
S WT A S T D N V - G V T G Y D V - Y
AWD A P S S G A - N I A E Y V V S F
S WA A A T G A L - P I A S Y T V - Y
T WK A S S D D T - G V T G Y D I - Y
TW
WQ A S S D D K - G V T G Y D V - Y
T WT A S T D E C S G V S G Y Q I - T
S WS A V S - - - - G A T K Y R V A Y
S WS A V S - - - - G A T K Y R V A Y
S WS A V S - - - - G A T K Y R V A Y
S WS A V S - - - - G A T K Y R V A Y
S WS A V S - - - - G A T K Y R V A Y
S WS A V S - - - - G A T K Y R V A Y
S WT A V S - - - - G A T S Y T I S Y
N I RT VKS KT -PVS KVE F -Y
K I T A T A S D S DG K I S R V D - F
70
R S S V E G G L 38
R - - S D G G T 29
R S S V E G G L 38
R - - S D G G T 29
Y
R S T V K G G L 38
K S L S E T G P 43
R S T K S G G P 38
Y
R - - - - D G - 38
V - - - - D G - 20
R - - - - D G - 38
N - - - - - G - 37
N - - - - - G - 37
E - - - - - - - 37
R - - - - N G - 38
A - - - - G G - 38
A - - - - D N - 38
R T G G T G G - 42
R - - - D A G 36
R - - - D A G 36
R - - - D A G 36
R - - - D A G 36
R - - - D A G 36
R - - - D A G 36
S - - T G S G A 37
A
N - - - - G D 50
46
L - - V DG
80
A I S A I DE L - - K V V AV DL S F NR
A I S AV DE L G NE
KVV - - - - - - - S V T A V D S DG N E
KVVA - - - - - - VVS AVS N - - - AV R AV DAA - - T V R A R DA AG NV
A V R A V DA AG NL
T I K A K DA AG NL
T V K A K DA AG NV
T V S A K D A DG K L
F V T A T DS QG NT
H V R A R D A A G NQ
V V R A K DA AG N I
T V K A K DG A G - K V R A E NS AG Y S
K V R A E NS AG Y S
K V R A E NS AG T S
K V R A E NS AG Y S
K V R A E NS AG Y S
K V R A E NS AG T S
K V R A E NS AG NS
A V A V L S DG R R I
I AY DDDDAAS T
- - - - - - - - - T E S NV V T I - S E MS I DT V A - - - - - - - - - S AL S NE V E A - - - - - - - - - - - - - - - - - - - - - - - - - - - S AAS APL T VT
S AT SGT VTGE
S AAS NAV T V S
S AAS NAV S V K
H AG P T V E V T S L PS S AL AVK
S ADS NT V - - S AP S NT V - - - - - - - - - - - A Y S AQ V DV T A Y S AQ V DV T AY S TQ V DV T A Y S AQ V DV T A Y S AQ V DV T AY S TQ V DV T AY - - - - - - - E S PVT PVL VK
P DS V K I F V KQ
- - - - - - - - - - - - - - - - - - - - - - - - - AR
- - - - - - - - - - - - - - - - - - - - - - - - - DV
K
74
77
86
61
86
76
75
71
66
84
84
84
81
84
82
82
76
83
83
83
83
83
83
76
98
98
Figure 3. Alignment of all 24 bacterial FNIII domains explicitly reported in literature [2,4,6,15,16,34,36,38-43], in carbohydrate acting
enzymes. Sequences were extracted as described in the Methods section, except for the two “FNIII-like domains” from C. thermocellum
CbhA (GenBank: CAA56918.1), which were not recognized as such by the domain annotation tools, and were therefore extracted manually
[15]. Predicted (Phyre2 [24]) conserved β-sheets are indicated in red. A conserved tryptophan in the 2nd β-sheet is indicated in blue, a conserved tyrosine in the 3rd β-sheet is indicated in pink, a conserved leucine is indicated in yellow, while a conserved aromatic residue in the
6th β-sheet is indicated in green (only the leucine residue is conserved in the two FNIII-like domains of CbhA). Other conserved residues are
indicated in grey. Amino acid numbering is based on MaAmyA-833.
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8 V. Valk, et al.
3.5 Enzyme activities associated with FNIII
domains
termini (Fig. 4). Interestingly, terminal FNIII domains are
found at a similar ratio at either terminus of the protein.
In contrast, most of the individual CBM families seem to
have a preference for either the N- or C-terminus (Fig. 4).
Taken together, these results show that FNIII domains
are mainly found in between other protein domains. This
supports the hypothesis that FNIII domains may function
as stable and/or flexible linkers.
Of all enzymes listed in CAZy, 2,486 proteins contain one or
more FNIII domains (Table 1). Of these 2,486 enzymes, 124
have been characterized and have at least one EC number
in the CAZy database. In Figure 5 an overview is shown of
the number of different EC numbers associated with these
80
70
60
50
Both
40
C-terminal
N-terminal
30
20
CBM15
CBM38
CBM37
CBM28
CBM40
CBM46
CBM45
CBM53
CBM17
CBM30
CBM54
CBM44
CBM58
0
FNIII
10
CBM
% proteins with domain at terminus
90
Figure 4. Percentage of FNIII or CBM containing proteins (excluding CBM68-CBM73) with at least one domain located at the terminal protein
end (defined as < 100 amino acid residues from the protein terminus, not containing any other defined domains). CBM: percentage of all
CBM containing proteins with a terminal CBM. For specific CBM families, the percentage of proteins with less than 50% terminal CBMs is
also shown.
20
15
Lyases
Hydrolases
10
Transferases
Oxidoreductases
5
0
FNIII
CBM1
CBM13
CBM32
CBM35
CBM2
CBM6
CBM20
CBM48
CBM3
CBM5
CBM16
CBM22
CBM4
CBM25
CBM10
CBM34
CBM50
CBM72
Number of unique EC numbers
25
Figure 5. Number of unique EC numbers associated with characterized FNIII and CBM containing proteins. Only CBM containing proteins
associated with at least five unique EC numbers are shown. The major EC activity groups are indicated: Oxidoreductases (EC 1.x), Transferases (EC 2.x), Hydrolases (EC 3.x), Lyases (EC 4.x). None of the proteins was associated with the Isomerase (EC 5.x) or Ligase (EC 6.x)
classes.
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enzymes. The enzyme classes, to which the EC numbers
belong, are also indicated. The same analysis was also
performed for the 50,301 CBM containing enzymes (Table
1), of which 2,273 have one or more EC numbers listed in
CAZy. CBM families associated with five or more different
EC numbers are included in Figure 5.
Enzymes that contain FNIII domains are associated
with 24 unique EC numbers (Table S1). In addition,
they are associated with all four main enzyme activities
listed in CAZy, more than for any of the CBMs (Fig. 5).
CBM1, CBM13 and CBM32 containing enzymes are
associated with 20 different EC numbers, the highest
number of unique EC numbers associated with a single
CBM family (Fig. 5). For individual CBM families it is
to be expected that they do not associate with a large
number of activities, since their main function is to bind
to specific substrates that also the associated catalytic
domain can interact with. For example, a CBM type
able to specifically bind cellulose would be of little use
in a starch degrading enzyme. This is, indeed, largely
reflected in the results on CBM association with EC
numbers (Fig. 5). Some CBMs are associated with a large
number of EC numbers, but in those cases the enzymes
mainly have activities related to the substrate that is
bound by the CBM or related substrates. We consider
a substrate related when in a natural environment it is
common for the carbohydrate that is bound by the CBM
to be in close proximity to the substrate of the catalytic
domain. For example, some xylanases have a cellulose
binding domain, but since both xylan and cellulose are
known to be in close proximity to each other in plant
cell walls [33] this is considered as a related CBM. FNIII
domains, on the other hand, are associated with many
different enzymatic activities, ranging from chitinacting (chitinase) and cellulose-acting (cellulose) to
starch-acting (α-amylase and pullulanase). Such a broad
distribution does not point to a role for FNIII as a binding
domain that supports interaction of the resident catalytic
domain with a specific substrate. The appearance of
FNIII domains in many different enzyme types instead
suggests a less specific role, e.g., as a stable linker.
3.6 FNIII domains function mainly as stable
linkers
Since 2002, three functions have been suggested for FNIII
domains in carbohydrate active enzymes [14-16]. This
paper shows that two previously characterized domains
of C. thermocellum cellobiohydrolase CbhA, in fact,
are not FNIII domains at all (Figs. 2, 3). Their proposed
function in substrate surface disruption thus no longer
FNIII domains in carbohydrate acting enzymes
9
holds for true FNIII domains. When FNIII and CBM
domains are compared for their position in the protein
and association with specific enzyme activities, a clear
difference is observed. While CBMs generally display a
certain substrate binding specificity and usually are part
of enzymes with specific catalytic activities for the same
substrate, this is not the case for FNIII domains. The
suggested role for FNIII domains in substrate binding
thus appears unlikely. It is well known that CBMs use
aromatic residues to interact and bind to carbohydrates.
However, the three-dimensional structure of the FNIII
domain from chitinase A1 showed that the domain did
not have any accessible aromatic residues [26], which
makes it less likely that it can function as a CBM. As
shown in Table 1, 71.7% of the FNIII containing proteins
also contain a CBM (as annotated by CAZy), which
increases up to 76.0% when dbCAN is used to annotate
the CBM domains. Finally, FNIII domains are mainly
located between other domains, unlike CBMs, which are
often present at the terminal end of proteins. On basis
of these considerations we conclude that the available
evidence suggests that FNIII domains function as stable
linkers in multi-domain proteins.
In truncation studies, the full-length enzyme is
usually compared with constructs, in which either the
CBM or both the CBM and FNIII domain(s) were removed.
In these studies, removal of only the CBM usually affects
activity on insoluble substrates [15,16,34-40], while the
additional removal of the FNIII domains showed no
further effects. In a study of the chitinase A1 from Bacillus
circulans WL-12a, the single CBM present was re-attached
after removal of one or two intermediate FNIII domains,
allowing a direct test for the FNIII function [14]. Removal
of one FNIII domain showed a ~20% activity decrease
on insoluble colloidal chitin, and removal of both FNIII
domains resulted in a ~50% activity decrease. Removal of
only the CBM also caused a ~50% activity decrease. When
the CBM was not re-attached, the removal of one or both
FNIII domains did not further affect activity. In all cases
the activity on a soluble substrate remained unaffected
[14]. These experimental data thus support the suggested
function of FNIII domains as stable protein linkers, acting
in the optimal placement of different enzyme domains
towards each other, and towards the substrate.
In conclusion, the multiple FNIII domains in the M.
aurum B8.A MaAmyA and MaAmyB proteins thus may act
as a spacer between the catalytic domain, the two CBM25
domains, and the CBM74 domain (in case of MaAmyA)
(Fig. 1), to provide optimal position and/or flexibility of
these domains with respect to each other and towards the
substrate.
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10 V. Valk, et al.
Acknowledgments: This study was partly funded by the
Top Institute of Food & Nutrition (project B1003) and by
the University of Groningen.
Conflict of interest: The authors declare no conflict of
interests.
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