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\begin{document}

\title{Structural elements determining the transglycosylating activity of
glycoside hydrolase family 57 glycogen branching enzymes}



\author[1]{Gang Xiang}%
\author[2]{Hans Leemhuis}%
\author[1]{Marc van der Maarel}%
\affil[1]{University of Groningen}%
\affil[2]{Avebe Group}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE)
catalyze the formation of an \selectlanguage{greek}α-\selectlanguage{english}1,6 glycosidic bond between \selectlanguage{greek}α-\selectlanguage{english}1,4 linked
glucooliogosaccharides. As an atypical family, a limited number of
GH57GBEs have been biochemically characterized so far. This study aimed
at acquiring a better understanding of the GH57GBE family by a
systematic sequence-based bioinformatics analysis of almost 2,500 gene
sequences and determining the branching activity of several native and
mutant GH57GBEs. A correlation was found between a very low or even no
branching activity with the absence of a flexible loop, a tyrosine at
the loop tip, and two \selectlanguage{greek}β-\selectlanguage{english}sheets.%
\end{abstract}%



\sloppy


\textbf{Structural elements determining the transglycosylating activity
of glycoside hydrolase family 57 glycogen branching enzymes}

Gang Xiang\textsuperscript{1}, Hans Leemhuis\textsuperscript{1,2}, and
Marc J.E.C. van der Maarel\textsuperscript{1}

1. Bioproduct Engineering, Engineering and Technology institute
Groningen (ENTEG), University of Groningen, Groningen, 9747 AG, the
Netherlands

2. Avebe Innovation Center, Groningen, 9747 AA, the Netherlands

Email address: Gang Xiang: g.xiang@rug.nl

Hans Leemhuis: hans.leemhuis@avebe.com

Marc J.E.C. van der Maarel: m.j.e.c.van.der.maarel@rug.nl

Corresponding author: Marc J.E.C. van der Maarel

\textbf{Abstract}

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE)
catalyze the formation of an \selectlanguage{greek}α-\selectlanguage{english}1,6 glycosidic bond between \selectlanguage{greek}α-\selectlanguage{english}1,4 linked
glucooliogosaccharides. As an atypical family, a limited number of
GH57GBEs have been biochemically characterized so far. This study aimed
at acquiring a better understanding of the GH57GBE family by a
systematic sequence-based bioinformatics analysis of almost 2,500 gene
sequences and determining the branching activity of several native and
mutant GH57GBEs. A correlation was found between a very low or even no
branching activity with the absence of a flexible loop, a tyrosine at
the loop tip, and two \selectlanguage{greek}β-\selectlanguage{english}sheets.

\textbf{Key words:} Glycogen branching enzymes, Glycoside hydrolase
Family 57, Glycogen, Amylose

\textbf{Introduction}

Glycogen branching enzymes (GBE) (EC 2.4.1.18) play a key role in the
biosynthesis of glycogen, a dendrimeric polyglucose carbon and energy
storage molecule present in many prokaryotic microorganisms, fungi,
yeast, and animals\textsuperscript{1-4}. GBE catalyze the formation of
\selectlanguage{greek}α-\selectlanguage{english}1,6 glycosidic linkages in glycogen by transglycosylating a
cleaved-off \selectlanguage{greek}α-\selectlanguage{english}glucan chain from a growing \selectlanguage{greek}α-\selectlanguage{english}1,4 linked
anhydroglucopyranose chain to the same or a different
chain\textsuperscript{1, 5-7}. In addition to the transglycosylating or
branching reaction, GBEs also catalyze the hydrolysis of an \selectlanguage{greek}α-\selectlanguage{english}1-4
glycosidic linkage using water as an acceptors\textsuperscript{8}, or
perform a cyclization reaction resulting in branched cyclic
glucans\textsuperscript{9}.

Based on the primary amino acid sequence and conserved motifs, all known
GBEs are categorized in either the glycoside hydrolase (GH) family 13 or
57\textsuperscript{10-13}. The GBEs from GH13 have a substantially high
activity on the model substrate amylose, a typical linear
\selectlanguage{greek}α-\selectlanguage{english}glucan\textsuperscript{14-17}. GH13 GBEs are involved in the classical
glycogen biosynthetic pathway by the tandem action of
glucose-1-phosphate adenylyltransferase (\emph{glgC} ) - glycogen
synthase (\emph{glgA} ) - glycogen branching enzyme (\emph{glgB}
)\textsuperscript{2, 18}. In contrast to GH13 GBEs, the role of GH57
GBEs is much less clear. So far, the biochemical properties of only five
GH57 GBEs have been reported, while the crystal structure of only four
of these five GH57 GBEs has been solved\textsuperscript{6, 10, 19, 20}.
The activity of these five GH57 GBEs on amylose is relatively low to
almost zero, ranging from a few mU/mg (\emph{Thermotoga maritima}SMB8)
to a few hundred mU/mg (\emph{Thermus thermophilus}
HB8,\emph{Thermococcus kodakarensis} KOD1)\textsuperscript{8, 21}. The
GH57 GBE of \emph{Mycobacterium tuberculosis} , having all the key
features of GH57 GBEs, was reported to have no detectable activity on a
range of \selectlanguage{greek}α-\selectlanguage{english}glucans\textsuperscript{22}. Based on the genomic
organization, it is assumed that this GBE branches glucosylglycerate and
plays a role in the biosynthesis of polymethylated
polysaccharides\textsuperscript{22-24}.

GH57 GBEs have a triangular three-dimensional shape consisting of three
domains including a catalytic (\selectlanguage{greek}β/α\selectlanguage{english})\textsubscript{7} barrel containing
the two catalytic residues, a glutamate nucleophile and an aspartate
acid/base catalyst\textsuperscript{6, 10, 19, 20}. All GH 57 GBEs have
five conserved sequence regions (CSR), with the nucleophile located in
CSRIII and the acid/base catalyst located in CSRIV\textsuperscript{25}.
Similar to GH13 GBEs, GH57 GBEs employ a double-displacement reaction
mechanism resulting in retention of the \selectlanguage{greek}α-\selectlanguage{english}configuration in the
products\textsuperscript{6}. In the crystal structures of the \emph{P.
horikoshii} , \emph{T. kodakarensis,} and \emph{T. thermophilus} GBE a
flexible loop with a conserved tyrosine at the tip was
identified\textsuperscript{6, 10, 19}. Mutational analysis showed that
the flexible loop and the tyrosine play a key role in the branching
activity; replacing the tyrosine with an alanine in the \emph{T.
thermophilus} GBE or shortening the loop in \emph{P. horokoshii} GBE
resulted in a loss of the branching activity\textsuperscript{6, 19}.

The simultaneous presence of a gene encoding a GH13 GBE and one encoding
a GH57 GBE in the genomes of a large number of bacteria makes the
physiological role of GH57 GBEs even more puzzling\textsuperscript{21}.
To gain more insight into the enzymatic activity and possible
physiological role of GH57 GBEs, an in-depth sequence-based
bioinformatics analysis of almost 2,500 GH57 GBE sequences and a basic
biochemical characterization of a number of carefully selected GH57 GBEs
overexpressed in \emph{Escherichia coli} was conducted. Surprisingly,
the flexible loop covering the active site was absent in the vast
majority of the GH57GBE sequences analyzed in this study. Several of
these loop-deficient GBEs displayed very low to no activity on amylose.
Besides the flexible loop an additional structural element, two adjacent
beta sheets, was identified to play a key role in the branching
activity. It is proposed that GH57 GBEs devoid of the flexible loop and
the two beta sheets are not glycogen branching enzymes and do not play a
role in glycogen biosynthesis but in one or more yet to be identified
metabolic pathways.

\textbf{2. Materials and methods}

2.1 Sequence collection, alignment and phylogenetic analysis

Sequences were collected by searching the key word ``DUF1957'' at the
National Center for Biotechnology Information (NCBI) on
04\textsuperscript{th} February 2020. To filter redundant and short
sequences, the source database and the sequence length were set at
RefSeq and 400-1,200 amino acid residues, respectively. Partial and
hypothetical sequences were deleted manually. Finally, 2,497 sequences
with the title ``DUF1957 domain-containing protein'' or ``Glycoside
hydrolase family 57 protein'' were used in the sequence alignment and
phylogenetic analysis. ClustalW multiple alignment was conducted with
the software package MEGA-X\textsuperscript{26} by setting gap opening
penalty at 10, the gap extension penalty at 0.2, the negative matrix at
off, and a 30 \% delay divergent cutoff. A phylogenetic tree was
constructed using the Jones-Taylor-Thornton (JTT) model with the maximum
likelihood method in default setting in the software package
MEGA-X\textsuperscript{26, 27}. Five conserved sequence regions were
analyzed by calculating possibilities of residues within conserved
sequence regions based on the multiple sequence alignment, as described
previously\textsuperscript{28}.

2.2 Identification of flexible loop and two beta sheets

Protein structures were visualized using the Pymol
software\textsuperscript{29}. The three-dimensional structures
of\emph{T. thermophilus} (PDB ID: 3P0B)\textsuperscript{6}, \emph{T.
kodakarensis} (PDB ID: 3N8T)\textsuperscript{10}, \emph{P.
horokoshii}(PDB ID: 5WU7)\textsuperscript{19} and \emph{T. maritima} GBE
(AmyC) (PDB ID: 2B5D)\textsuperscript{20} were downloaded from the
Protein Data Bank (PDB). The distances between two residues was measured
with the measurement command in Pymol. The flexible loop and the two
beta sheet regions were defined based on the crystal structures.

2.3 Overexpression and enzyme production

Various putative GH57 GBEs were overexpressed in \emph{Escherichia
coli}(Table 1), and several mutants were also constructed (Table 2).
Genes with optimized codons for overexpression in \emph{Escherichia
coli} were synthesized and then ligated in the pRSET B (\emph{T.
kodakarensis} GBE,\emph{T. thermophilus} GBE and \emph{T. maritima} GBE)
or pET 28a(+) (the remaining GBEs used in this study) vector by
Genscript (USA). Plasmids were transformed into \emph{E. coli} BL21
(DE3) by subjecting competent cells to a heat shock at
42\textsuperscript{o}C for 90 seconds. A single colony was selected from
ampicillin or kanamycin-containing agar plate for further cultivation.
\emph{E. coli}strains carrying the target gene were stored in 25\%
glycerol stock at -80\textsuperscript{o}C. \emph{E. coli} BL 21(DE3)
carrying the native or mutant \emph{gbe} was grown in Luria-Bertani (LB)
medium (Becton, Dickinson and Company, USA) at 37\textsuperscript{o}C
and 150 rpm. When the optical density reached 0.6-0.8 (600 nm), protein
expression was induced at 20\textsuperscript{o}C for 12 hours by adding
Isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG) (Thermo Fisher Scientific,
USA) to a final concentration of 0.2 mM. In order to enhance the
solubility of the target protein, the chaperone plasmid pG-KJE8 (Takara,
Japan) was co-expressed in \emph{E. coli} by following the manufacture's
protocol. Cells were harvested by centrifugation at 8,000\selectlanguage{ngerman}×\emph{g} for
10 min at 4\textsuperscript{o}C, washed cells twice with 50 mM phosphate
buffered saline (PBS) buffer (pH 7.0), and finally resuspended in 5 mM
PBS (pH7.0). Cells were disrupted by high pressure homogenizer (Avestin,
Canada) at room temperature, with an air pressure of 8 bar and a valve
pressure of 6 bar. Heat treatment was performed at
50\textsuperscript{o}C (\emph{Calidithermus timidus}
GBE,\emph{Meiothermus silvanus} GBE, \emph{Petrotoga mexicana} GBE) or
65\textsuperscript{o}C (the remaining GBEs used in this study) for 15
minutes to denature \emph{E. coli} host proteins. Then the supernatant
was incubated with nickel metal resin, followed by removal of
non-specifically bound host protein by applying a washing buffer
containing 25 mM imidazole (Sigma-Aldrich, USA) and elution of the
target protein with 250 mM imidazole. Imidazole was removed using a
desalting column (Thermo Fisher Scientific, USA) and a storage buffer to
5 mM PBS (pH7.0). Purity of the target protein was checked by SDS-PAGE.
Protein concentration was assayed by Bradford (Bio-Rad, USA) using
bovine serum albumin as standard.

2.4 Determination of catalytic activity

The branching reaction was performed at 50\textsuperscript{o}C in 5 mM
PBS buffer (pH7.0) using 0.125\% amylose V (Avebe, The Netherlands) as
substrate, and a final concentration of enzyme ranging from 0.03 mg/ml
to 0.13 mg/ml was used depending on the total activity of enzyme.
Samples were taken at time intervals to monitor the reaction progress.
The reaction was stopped by boiling samples at 100\textsuperscript{o}C
for 10 min. Branched samples were treated with \emph{Pseudomonas} sp
isoamylase (0.4 U/ml) and \emph{Klebsiella planticola} pulullanase M1
(1.4 U/ml) (Megazyme, Ireland) at 40\textsuperscript{o}C for 24 h with
constant mild shaking. Reducing ends were quantified by the
2,2'-bicinchoninic acid (BCA) method using glucose as
standard\textsuperscript{30}. The activity unit was calculated based on
reducing end profiles of samples before and after debranching. One unit
of activity is defined as 1 \selectlanguage{greek}μ\selectlanguage{english}mol reducing ends released or transferred
per minute under aforementioned reaction conditions.

\textbf{3. Results and discussion}

\textbf{3} .\textbf{1 Sequence-based analysis of putative GH57 GBE
sequences}

Using the key words ``DUF1957 domain-containing protein'' or ``Glycoside
hydrolase family 57 protein'', 2,497 amino acid sequences were retrieved
from the NCIB database. These sequences varied in length between 418 and
1,184 amino acids. Except for 50, all sequences had the nucleophile and
acid/base catalyst and contained the five conserved sequence regions
typical for GH57 members (Fig. 1). The exception were sequences that
missed one or both catalytic residues and showed a large variation in
four of the five conserved sequence regions. These sequences were
excluded from further analysis as it was assumed that they are not
active. The first four conserved sequence regions are positioned within
the A-domain containing the (\selectlanguage{greek}β/α\selectlanguage{english})\textsubscript{7} barrel. Conserved
region 5 is located in the C domain on the second \selectlanguage{greek}α \selectlanguage{english}helix.

When comparing the sequence logo for all the GH57 GBEs of this study
with the logos recently published on 1,602 GH57
sequences\textsuperscript{25}, two GBE specific fingerprints become
clear; the first is a quintet of amino acids with the combination HxHLP,
with x being A, S or T, found in CSRI of almost all GBE sequences; in a
small number of GBE sequences the L at position 4 is replaced by an I or
M. In all other GH57 enzymes a Q is present instead of an L at position
4 whereas in \selectlanguage{greek}α-\selectlanguage{english}galactosidases and -related proteins there is an L at
position 4 but this is followed by a Q or A/M and not a P as is the case
for GH57 GBEs. The second GBE fingerprint is the sextet ELF(Y)GHW
present in CSRIV. The first position of this fingerprint, the E, is
conserved among all proteins assigned to a functional GH57 enzyme
subfamily. This E is not conserved in the proteins that are categorized
as -like proteins. These proteins miss one or both of the catalytic
residues and are very likely not active. In the 4-\selectlanguage{greek}α-\selectlanguage{english}glucanotransferase
of \emph{Thermococcus litoralis} the E is only 5.1 \selectlanguage{ngerman}Å from the acid-base
catalyst D (Fig. 2A) and is involved in binding the -1 subsite residue
through a water molecule\textsuperscript{31}. In the other GH57 crystal
structures, the conserved E is 4 Å (\emph{T. maritima} AmyC) to 7 Å
(\emph{T. thermophilus} GBE) from the acid-base catalyst (Fig. 2B and
2C). In GH13 enzymes, a catalytic triad of a catalytic nucleophile D, a
general acid base catalyst E, and a transition state stabilizer D play a
key role in catalysis {[}32-34{]}. As the CSRIV E is completely
conserved in all GH57 proteins assigned to a functional subfamily and is
positioned close to the acid-base catalyst in all available GH57 crystal
structures, it is not unlikely to assume that this E plays a similar
role as the transition state stabilizer D in GH13.

The other positions of the sextet are completely conserved in all GBE
proteins analyzed in this study, with the exception of the third
position, the F which is in \emph{K. pacifica} GBE replaced by another
hydrophobic side chain containing amino acid, an Y. Whereas previously
it was reported that the C at position 16 (CSRIII) is conserved among
GH57 GBEs\textsuperscript{6}, this position is not absolutely invariant,
as 72 out of the 2,497 (2.9\%) sequences have a different amino acid in
this position, a feature also noticed by Martinovi\selectlanguage{polish}č\selectlanguage{english}ov\selectlanguage{ngerman}á and
Jane\selectlanguage{polish}č\selectlanguage{english}ek\textsuperscript{25}; the majority of these have an M (56;
2.2\%), nine have an S, five an L and two an F. This almost fully
conserved C can still be seen as a fingerprint as none of all the other
GH57 enzymes and -like proteins have a C at this position.

In addition, five residues in the vicinity of the active sites were
identified to be fully conserved in all sequences; three tryptophans
(W274, W404 and W413), one histidine (H146), and one arginine (R265)
(\emph{T. thermophilus} numbering). In \emph{T. kodakarensis} , the
three tryptophans and the one at position 28 have been defined as the
aromatic gate keepers\textsuperscript{8}. This group of four aromatic
gate keeper tryptophans is highly conserved in all GH57 GBEs except W28,
which is a threonine in the GBEs of \emph{Thermus} and
\emph{Meiothermus} species. In the \emph{T. thermophilus} GBE the W274
is positioned to the side and the W404 at the bottom of the positive
subsites\textsuperscript{6}. Both are involved in substrate binding by
aromatic stacking (W274) and hydrogen bonding (W404). In the \emph{T.
maritima} GBE, the W274 equivalent (W246) is buried such that aromatic
stacking is very unlikely to occur while the position of the W413
equivalent (W411) is difficult to predict\textsuperscript{6}. The role
of the H146 and the R265 is not clear.

In the \emph{P. horokoshii} GBE, a tryptophan (W22) at the bottom of the
active site groove is involved in substrate recognition. Changing this W
into an A resulted in almost complete loss of
activity\textsuperscript{19}. This W is also found at the same position
in the crystal structure of \emph{T. thermophilus} and \emph{T.
kodakarensis} . In GH57GBEs from \emph{T. maritima} , \emph{P. mexicana}
,\emph{P. mobilis} and \emph{K. pacifica} , this W is replaced by D or E
or P. Besides the bottom W four other aromatic amino acids are found in
close vicinity of the active sites of \emph{T. kodakarensis} , \emph{T.
thermophilus} , \emph{P. horokoshii} , or \emph{T. maritima} ; F23,
F289, W360 and F461 (\emph{T. thermophilus} numbering). In all the other
sequences of this study, three of these four aromatic amino acids are
functionally conserved while the F23 is not conserved. Zhang et
al.\textsuperscript{8} reported another three important amino acids near
the active site, H11, S462, and D463 (\emph{T. thermophilus} numbering).
These are all conserved at the respective positions in all the 2,497
sequences.

\textbf{3.2 The flexible loop and tyrosine}

From the sequence alignment, two major gaps became visible. The first
was a sequence of 19 amino acids from position 229 to 247 (\emph{T.
thermophilus} GBE numbering) in between CSR III and IV. In the crystal
structures of \emph{T. thermophilus} \textsuperscript{6}, \emph{T.
kodakarensis} \textsuperscript{10}, and \emph{P. horokoshii}
\textsuperscript{19} GBE, these amino acids make up a flexible loop
covering the catalytic cleft (Fig. 3). In 201 other amino acid
sequences, a flexible loop of varying length is present (Fig. 4). The
average loop size is 26 amino acids with the shortest being 13 amino
acids, while the longest is 50 amino acids. At the tip of the flexible
loop of \emph{T. thermophilus} , \emph{T. kodakarensis} , and \emph{P.
horokoshii} GBE, a tyrosine is present\textsuperscript{6, 10, 19}.
The\emph{T. thermophilus} tyrosine was shown to play a prominent role in
the branching activity. Replacing this tyrosine with an alanine resulted
in a loss of branching activity\textsuperscript{6}. A minority of GBEs
with a flexible loop do not have a tyrosine at the tip (35; 17.2\%).
Instead an alanine, serine, or threonine are found.
The\emph{Thermoanaerobaculum aquaticum} GBE, which has a medium sized
loop of 22 residues with an alanine at the tip, has a relatively high
activity towards amylose, dominated by the branching activity of 168
mU/mg and a ratio of branching over hydrolytic activity of 38.1 (Table
3). On the contrary, the \emph{Calidithermus timidus} GBE with a
flexible loop of 23 amino acids and a tyrosine at the tip, has a
branching activity of 356.9 mU/mg, being twice that \emph{T.
aquaticum}GBE. This result indicates that the tyrosine at the tip of the
flexible loop does not play a determining role in the branching
activity. This was confirmed by a tyrosine to alanine mutation in the
\emph{T. kodakarensis} GBE, which has a flexible loop of 19 amino acids,
and the same size and configuration as that of the \emph{T.
thermophilus} GBE (Table 3; Fig. 3). Whereas the wild type \emph{T.
kodakarensis} GBE has a dominant branching activity of 480 mU/mg and a
branching over hydrolysis ratio of 41, the Y/A mutant retains the
dominant branching activity (426 mU/mg). The hydrolytic activity of the
Y/A mutant doubled from 12.6 mU/mg to 26.3 mU/mg, resulting in a
branching over hydrolysis ratio of 16.2. These results suggest that not
only the tyrosine at the tip of the loop but also the size and
configuration of the flexible loop play a role in the branching
activity.

In contrast to the \emph{T. thermophilus} , \emph{T. kodakarensis}
and\emph{P. horokoshii} GBE, the flexible loop is absent in the \emph{T.
maritima} GBE (Fig. 3 and 5), as was already noted by Zhang and
coworkers\textsuperscript{8}. It has been reported that AmyC, in spite
of the lack of a flexible loop, has a low but reproducible branching
activity towards amylose\textsuperscript{8}. Shortening of the flexible
loop in the \emph{P. horokoshii} GBE from 19 to 9 amino acids resulted
in a twofold increase in total activity and a considerable reduction of
the branching activity\textsuperscript{19}. The flexible loop is not
only absent in AmyC but also in 2,296 of the 2,497 sequences (92\%) used
in this study (Table 4). A comparable low branching activity was found
for the GBE of \emph{P. mexicana} and \emph{Kosmotoga pacifica} , of
14.1 mU/mg and 9.1 mU/mg respectively (Table 3). Although having a low
activity, both enzymes introduced 6\% branches in the final product when
incubated with amylose V (data not shown), confirming that both enzymes
clearly have branching activity. Introduction of the full or partial
flexible loop of \emph{T. kodakarensis} GBE, including the tyrosine, in
the \emph{T. maritima} GBE resulted in a two-fold increase of the
branching activity and a three-fold increase of the hydrolytic activity
(Tables 3), being substantially lower than the activity of the wild
type\emph{T. kodakarensis} GBE. The flexible loop appears not to be the
only structural element that determines the overall activity of the GH57
GBEs.

The length of about 19 residues and configuration of the \emph{T.
thermophilus} and \emph{T. kodakarensis} GBE flexible loop seems to be
optimal with respect to the branching activity. Hundred and three of the
201 sequences (51\%) have a flexible loop of 17 to 22 amino acids (Fig.
4). A smaller, but still significant number of proteins (81; 40\%) have
a flexible loop of 24 or more amino acids while 17 proteins (9\%) have a
flexible loop of 13 to 15 amino acids. The models of the \emph{T.
hugenholzii} and \emph{T. lipolytica} GBE, with 36 and 28 amino acids
loop resp., show that only the first part of the flexible loop folds
into the active site cleft while the rest folds next to or behind the
part that covers the cleft (Fig. 5). The \emph{T. hugenholzii} flexible
loop contains a tyrosine at position 245 which is turned away from the
cleft being far away from the two active site residues (E191 and D382).
This GBE has a very low branching activity of only 13.9 mU/mg, while
the\emph{T. lypolytica} GBE did not show any activity (Table 3). Thus,
for a GH57 GBE to act as a ``true'' glycogen branching enzyme, a
flexible loop of 17-22 amino acids covering the active cleft and, when
present, a tyrosine positioned deep into the active site close to the
catalytic E and D is required. The absence of the flexible loop or a
loop smaller or larger than 17-22 amino acids results in a significant
reduction to complete loss of activity towards amylose. What the
\emph{in-vivo}substrate for these loop-deficient and long-looped GH57
GBEs is, remains to be established. It could be that these GBEs do show
activity towards a growing \selectlanguage{greek}α-\selectlanguage{english}glucan chain, the \emph{in-vivo} substrate
of most GBEs\textsuperscript{1}.

\textbf{3.3. The two beta sheets}

The second gap in the sequence alignment is in the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region, where
in the \emph{T. thermophilus} GBE two prominent beta sheets (\selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6;
188-205, \emph{T. thermophilus} GBE numbering), are
localized\textsuperscript{6}. Without exception, all 2,296 GBEs (92 \%)
that miss the flexible loop also do not have the \selectlanguage{greek}β\selectlanguage{english}5 nor \selectlanguage{greek}β\selectlanguage{english}6 sheet (Table
4). In contrast, the majority of the 201 sequences that have a flexible
loop also possess the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region (159 or 79.1\%). Four of the GBEs
with a flexible loop and the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region were included in this study
and showed considerable branching activity ranging from 168 mU/mg to 550
mU/mg. Of those GBEs with a flexible loop but without the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region,
one, that from \emph{Meiothermus} sp. PNK-Is4, was overproduced and
analysed. It was active towards amylose but as other GBEs without the
two beta sheets, it only had a very low branching activity (29.1 mU/mg).
Also, the branching over hydrolysis ratio was very similar to those GBEs
that missed the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region; e.g. \emph{K. pacifica} and AmyC with a
branching over hydrolysis ratio of 3.8 and 5.7 respectively. A similar
result was found for the \emph{T. hugenholzii} GBE, having a long
flexible loop of 36 amino acids with a tyrosine and a very short \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6
region (Table 3). No activity could be detected for the \emph{T.
lipolytica} GBE, which has a long flexible loop of 28 residues without a
tyrosine and no \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region. Removing the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region from the\emph{T.
kodakarensis} GBE resulted in a complete loss of activity (Table 3).

Interestingly, the \emph{Mycobacterium tuberculosis} H37Rv GH57 GBE also
misses the flexible loop as well as the \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region. Gusthart
overexpressed the \emph{M. tuberculosis} GH57GBE but did not find any
activity\textsuperscript{22}. When incubating the \emph{M.
tuberculosis}GH57GBE with amylose, no activity was detected (Gang and
van der Maarel, unpublished results). The \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 region thus plays a
determining role in the activity of GH57 GBEs, in particular in the
branching activity. How exactly this beta sheet region exerts its
influence on the branching activity remains unclear.

\textbf{4. Conclusions}

Bioinformatics analysis reveals the sequence-based features of putative
GH57GBE sequences, that are two GH57GBE-specific sequence logos,
variations in flexible loop and presence/absence of two \selectlanguage{greek}β-\selectlanguage{english}sheets, were
noted in 2,497 sequences, with a majority of them (92\%) having no
flexible loop and absence of two \selectlanguage{greek}β-\selectlanguage{english}sheets; Biochemical data verified
that putative GH57GBE sequences can be defined by the former feature and
true GH57GBEs can be designated by the latter two structural features
which were assigned as optimal flexible loop and presence of two
\selectlanguage{greek}β-\selectlanguage{english}sheets. These results guide us to further investigate GH57GBEs or
--like sequences in an explicit direction.

\textbf{Acknowledgments}

We thank China Scholarship Council (GX) and University of Groningen for
financial support. Special appreciation to Zhenhua Zhang for assistance
with sequence collection.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interests.

\textbf{Data availability statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{References}

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

* Two catalytic residues are indicated by a red star symbol.

Figure 1. Fingerprint logos of five CSRs among 2,447 glycoside hydrolase
family 57 glycogen branching enzyme homologues\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Distance in \selectlanguage{ngerman}Å between the conserved E of CSRIV (blue) and
catalytic residues E in CSR III and D in CSR IV (red). A. GH57
4-\selectlanguage{greek}α-\selectlanguage{english}glucanotransferase of \emph{Thermococcus litoralis} ; B. GH57 AmyC
of\emph{Thermotoga maritima} ; C. GH57 Glycogen branching enzyme
of\emph{Thermus thermophilus} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3. Partial alignment of the regions containing the \selectlanguage{greek}β\selectlanguage{english}5 and \selectlanguage{greek}β\selectlanguage{english}6
sheets and the flexible loop plus tyrosine of the five biochemically
characterized glycoside hydrolase family 57 glycogen branching enzymes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4. Length distribution of the flexible loop among the 201
loop-containing glycoside hydrolase family 57 glycogen branching enzymes\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5. Configuration of the several glycoside hydrolase family 57
glycogen branching enzyme showing the position of the flexible loop, the
tyrosine at the tip of the flexible loop, and the presence/absence of
the \selectlanguage{greek}β\selectlanguage{english}5 and \selectlanguage{greek}β\selectlanguage{english}6 sheets (purple). The insert shows the position and
distance of the tyrosine at the tip of the flexible loop and the two
catalytic residues.

Table 1. Information and sequence similarity of various putative
GH57GBEs used in this study.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Organism\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Abbreviation\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Accession number\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Length (aa)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Sequence identity (\%)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermus thermophilus} HB8\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Tt GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_011228999.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
520\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100.00\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Calidithermus timidus} DSM 17022\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ct GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_018467494.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
708\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
66.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Meiothermus} sp. PNK-Is4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ms GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_129865543.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
520\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
62.9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermoflexus hugenholtzii} JAD2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Th GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_088572346.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
558\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
46.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermococcus kodakaraensis} KOD1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Tk GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_011250387.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
675\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
44.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermoanaerobaculum aquaticum} MP-01\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ta GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_053334947.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
522\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
39.4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermosyntropha lipolytica} DSM 11003\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Tl GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_073092191.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
519\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36.4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Kosmotoga pacifica} SLHLJ1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Kp GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_047754759.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
531\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Petrotoga mexicana} DSM 14811\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Pme GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_103077822.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
538\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
33.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Thermotoga maritima} SMB8\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Tm GBE\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
WP\_004081707.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
528\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
32.8\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 2. Mutants of tyrosine residue, flexible loop and two beta sheets
used in this study\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Mutant enzyme code & Mutant type & Sequence\tabularnewline
Tk GBE Y233A & Single site mutation & 221 SRLID
EGPASNV\emph{A}GEVLIADTEKT TLRP248\tabularnewline
Tm GBE LS & Loop swap & 208 SHAFW \emph{EGPASNVYGEVLIADTEKT}
GVYR236\tabularnewline
Tm GBE LE & Loop extension & 208 SHAFW FADEQPRY\emph{GEVLIADTEKT}
GVYR236\tabularnewline
Tk GBE \selectlanguage{greek}β \selectlanguage{english}sheet deletion & \selectlanguage{greek}β \selectlanguage{english}sheet deletion & 182 PECAY
\textbackslash{}soutRPAGEWELPGGRKVKR QGIE190\tabularnewline
\bottomrule
\end{longtable}

Table 3. Branching and hydrolytic activity of various glycoside
hydrolase family 57 glycogen branching enzymes. The activity was
determined using amylose as substrate.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Structural element}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Structural element}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Structural element}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Branching} \textbf{(mU/mg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Branching} \textbf{(mU/mg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Hydrolytic} \textbf{(mU/mg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{B/H\textsuperscript{\&}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{Loop}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{Tyrosine}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{β sheets}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{Wild-type enzyme}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermus thermophilus} HB8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
480.0 \selectlanguage{ngerman}± 3.5\textsuperscript{\#}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
480.0 \selectlanguage{ngerman}± 3.5\textsuperscript{\#}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
11.7 \selectlanguage{ngerman}± 1.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
41.0 \selectlanguage{ngerman}± 2.1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermococcus kodakaraensis} KOD1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
550.0 \selectlanguage{ngerman}± 5.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
550.0 \selectlanguage{ngerman}± 5.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
12.6 \selectlanguage{ngerman}± 1.7\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
43.6 \selectlanguage{ngerman}± 3.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Calidithermus timidus} DSM 17022\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
356.9 \selectlanguage{ngerman}± 10.6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
356.9 \selectlanguage{ngerman}± 10.6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
15.3 \selectlanguage{ngerman}± 2.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
24.1 \selectlanguage{ngerman}± 4.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermoanaerobaculum aquaticum} MP-01\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
168.0 \selectlanguage{ngerman}± 9.8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
168.0 \selectlanguage{ngerman}± 9.8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.4 \selectlanguage{ngerman}± 0.2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
38.3 \selectlanguage{ngerman}± 4.1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Meiothermus} sp. PNK-Is4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
29.1 \selectlanguage{ngerman}± 3.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
29.1 \selectlanguage{ngerman}± 3.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.5 \selectlanguage{ngerman}± 1.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.7 \selectlanguage{ngerman}± 1.7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermosyntropha lipolytica} DSM 11003\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\textsuperscript{\$}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
/\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermoflexus hugenholtzii} JAD2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
13.9 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
13.9 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.5 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
30.6 \selectlanguage{ngerman}± 3.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Petrotoga mexicana} DSM 14811\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
14.1 \selectlanguage{ngerman}± 3.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
14.1 \selectlanguage{ngerman}± 3.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.4 \selectlanguage{ngerman}± 0.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
10.0 \selectlanguage{ngerman}± 0.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Kosmotoga pacifica} SLHLJ1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
9.1 \selectlanguage{ngerman}± 0.8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
9.1 \selectlanguage{ngerman}± 0.8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.4 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
3.8 \selectlanguage{ngerman}± 0.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Thermotoga maritima} SMB8\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{+}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
10.5 \selectlanguage{ngerman}± 0.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
10.5 \selectlanguage{ngerman}± 0.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.8 \selectlanguage{ngerman}± 0.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 0.1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{Mutant enzyme}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Tk GBE Y233A\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
426.2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
26.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
26.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
16.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Tm GBE LS\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
24.4 \selectlanguage{ngerman}± 3.9\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.2 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.2 \selectlanguage{ngerman}± 0.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.0 \selectlanguage{ngerman}± 0.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Tm GBE LE\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
22.6 \selectlanguage{ngerman}± 3.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.6 \selectlanguage{ngerman}± 0.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.6 \selectlanguage{ngerman}± 0.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
4.0 \selectlanguage{ngerman}± 0.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Tk GBE \selectlanguage{greek}β \selectlanguage{english}sheet deletion\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
/\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{\&} ratio branching activity to hydrolysis activity

\textsuperscript{\#} average of three independent measurements with
standard deviation

\textsuperscript{\$} NA = no activity

Table 4. Distribution of flexible loop and \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 sheets among 2.497
glycoside hydrolase family 57 glycogen branching enzymes\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Structural elements & \# sequences & \% total\tabularnewline
\midrule
\endhead
No loop and no \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 sheets & 2,296 & 92\tabularnewline
Loop and \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 sheets & 159 & 5.3\tabularnewline
Loop and no \selectlanguage{greek}β\selectlanguage{english}5-\selectlanguage{greek}β\selectlanguage{english}6 sheets & 42 & 2.7\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

