Escolar Documentos
Profissional Documentos
Cultura Documentos
to enhance glycodiversification
a
a
a
David Daude
, Isabelle Andre
, Pierre Monsan and
b,c
Magali Remaud-Sime
on*
DOI: 10.1039/9781849739986-00624
Carbohydrates are biomolecules that have an essential role in every form of life. The
reservoir of naturally occurring glyco-structures is incredibly large and involves a tremendous number of carbohydrate-active enzymes (more than 280,000 released modules
in the Carbohydrate Active enZymes database) for their synthesis and degradation.
Nevertheless, natural enzymes do not necessarily present all the requested properties in
terms of eciency, specificity or stability when considering their usage for carbohydrate
or glyco-derivative manufacturing. In addition, if existing, the identification of an enzyme
perfectly adapted to a specific function from the natural diversity may be critical due to the
lack of available biochemical data and may necessitate intensive screening eorts. To
circumvent such limitations and provide optimized solutions, protein engineering has
been considered. Leloir-type glycosyltransferases, for example, are mainly involved in the
biosynthesis of glycoconjugates in Nature and they have been widely studied and engineered for this purpose. However, these enzymes are often found as membrane-bound
proteins, what renders dicult their isolation and purification. In addition, their need of
low-abundant activated sugars as glycosyl donors also impairs their usage. Alternatively,
enzymes that use more abundant glycosyl donor directly issued from agro-ressources
have been considered to access to new glyco-derivatives. This has promoted the use of
glucansucrases (GS) that catalyze transglycosylation reactions from sucrose substrate.
These enzymes are of particular interest for synthetic purpose and have found industrial
interest for pharmaceutical and fine chemical applications. To diversify their applications,
various approaches of engineering have been exploited to improve expression level,
stability, or change substrate or product specificity of these enzymes. In particular, the
range of molecules recognized and the osidic linkages formed by GS is broad but yet
limited. Therefore, protein engineering methods have been applied to further increase the
diversity of glycosylation reactions catalyzed by these enzymes. Sequence analysis and
mutagenesis experiments have enabled the identification of key amino acid residues of
glucansucrases either involved in catalysis or substrate specificity. Moreover, the determination of three-dimensional structures of glucansucrases from both families 13 and
70 of Glycoside-Hydrolases (GH) have provided powerful information for understanding
the sequence-structure-function relationships and guiding structure-based rational and
semi-rational engineering of these proteins. To assist these eorts, high-throughput
screening and biomolecular methods have been developed for the directed evolution of
these enzymes. Here are reported some of the successes in the bioengineering of glucansucrases from precursor work to latest results, as well as the methods developed for
screening and developing ecient variant libraries. The major progresses and breakthroughs in the field will be highlighted and further prospects will be considered and
discussed.
Introduction
Glucansucrases (GS) are a-transglucosylases involved in a-glucan biosynthesis. These enzymes have been classified as glycosyltransferases by
the Enzyme Commission (EC 2.4.1.4, EC 2.4.1.5, and EC 2.4.1.140) and
placed in the category of Glycoside-Hydrolases (GH) by the CAZy classification according to their sequence and structure similarities.1 Most
glucansucrases (dextransucrases, alternansucrases, reuteransucrases and
mutansucrases) belong to the GH70 family and synthesize a-glucans
harbouring various osidic linkages (Fig. 1B) with the exception of
amylosucrases, responsible for the synthesis of an amylose-like polymer,
which are part of the GH13 family. Unlike Leloir-glycosyltransferases,
glucansucrases do not require activated nucleotide-sugars as donor
substrates as they use sucrose, which contains an osidic linkage with an
energetic level similar to the one of nucleotide-sugars. From sole sucrose,
GS naturally catalyze the synthesis of a-glucans, as well as the glucosylation of hydroxylated acceptors such as gluco-oligosaccharides, sucrose
or fructose (Fig. 1B). They follow an a-retaining mechanism involving
first the formation of a b-D-glucosyl-enzyme covalent intermediate with a
concomitant release of fructose (Fig. 1A). In a second step, this intermediate is attacked by the hydroxyl group of an acceptor to release the
glucosyl residue. Glucan synthesis occurs via successive transfers of the
glucosyl units onto glucooligosaccharides. Depending on the enzyme
specificity, the produced polymers dier in terms of size as well as
number, type and arrangement of osidic linkages.
The scope of reactions catalyzed by glucansucrases can also be extended by introducing exogenous hydroxylated molecules into the reaction media which, if recognized by the enzyme, can play the role of
acceptor in glucosylation reactions. A variety of oligosaccharides or glucoconjugates can thus be synthesized depending on the nature of the
considered acceptor and the enzyme specificity. A wide range of acceptors has been reported for GS belonging to GH70 family and the main
ones are shown in Fig. 2. Molecules from various types can be recognized
by glucansucrases, ranging from monosaccharides structurally similar to
the natural acceptor to unconventional substrates such as amino acid
derivatives or bulky flavonoids. Amylosucrases from GH13 family are also
known to glucosylate diverse acceptors, albeit they have been less investigated for this purpose (Fig. 3). Though several acceptor substrates
have been identified, only a small number of donors have been reported
to date. Fluoro-glucosides, p-nitrophenyl-a-D-glucopyranoside and maltooligosaccharides have been shown to act as glucosyl donors for amylosucrases.24 Interestingly, some sucrose derivatives harbouring a
modified glucosyl moiety have been described as possible substrates for
transglycosylation reactions. The 2-ketosucrose for example has been
identified as an alternative donor substrate of glucansucrases for synthesizing carbonyl-group-containing dextran.5 The a-D-galactopyranosyl1,2-b-D-fructofuranoside and allo-sucrose have also been mentioned as
potential donors for amylosucrases, even if further characterization may
be required.6,7 Nevertheless, most sucrose derivatives and analogs
Carbohydr. Chem., 2014, 40, 624645 | 625
OR
-O
glucosylation
O
H
O
O
11
13
Sucrose
(12)
Hydrolysis
O
-O
15
Leucrose
Turanose
O
H
Glucose
12
16
Fructose
16
14
16
14
16
16
13
16
13
13
16
Polymerization
14
13
16
16
12
16
14
13
16
16
14
HO
OR
-O
O
Non-saccharidic acceptor
Reuteran
Mutan
Dextran
Alternan
Amylose
R = Fructose
2
R = H : Hydrolysis
2
R = Carbohydrate : Transglucosylation
2
R = Fructose : Sucrose isomerization
deglucosylation
Monosaccharide
16
16
13
16
13
14
Transition state
14
GH70 products
14
GH13 product
Trehalulose
R2OH R1OH
Sucrose isomerization
Transition state
Fig. 1 Reactions catalyzed by glucansucrases. (A) General mechanism; (B) Overview of the products generated by glucansucrases.
Glucoconjugates
Acceptor reactions
Oligosaccharides
HO
OH
OH
O
HO
HO
H 3C
HO
OH
HO
NHAc
N-Acetyl-D-glucosamine
HO
HO
OH
OH
-D-Tagatose OH
1,5-Anhydro-D-fructose
[41]
[42]
OH
HO
OH
OH
OH
Methyl -D-Glucopyranoside
OCH 3
OH
Methyl -D-Glucopyranoside
L-Glucose
[43]
HO
HO
OH
OH
OCH 3
[43]
OH
O
HO
HO
OH
O
HO
HO
OH
L-Rhamnose
[41]
OH
O
OH
[44, 45]
HO OH
OH
HO OH
HO
OH
OCH 3
HO
OCH 3
HO
OCH 3
OH
Methyl -D-Galactopyranoside
Methyl -D-Mannopyranoside
Methyl -D-Galactopyranoside
[44]
[44]
[44]
OH
OH
OH
HO
OH
O
HO
HO
OH
O
HO
HO
OH
OCH 3
OH
Methyl -D-Mannopyranoside
OH
Methyl -D-Allopyranoside
[44]
OCH 3
OH
OCH 3
Methyl -D-Allopyranoside
[44]
[44]
OH
OCH 3
H 3C
HO
HO
HO
OH
O(CH 2 ) n CH 3
HO
OH
Methyl -L-Rhamnopyranoside
Alkyl -D-Glucopyranoside
[41]
n=0, 3, 7...
[47]
OH
OH
HO
HO
HO
HO
OH O
OH
OH
O
O
HO
OH
Maltose
HO
HO
OH
[233, 236-238]
OH
OH
Isomaltose
[45, 48]
OH
HO
HO
HO OH
O
OH
O
OH
HO
OH
Gentiobiose
[49]
HO
OH
OH
HO
HO
OH
OH
Lactose
[50]
OH
HO
HO
HO
HO
HO
OHOH
O
OH
OH
OH
O
O
OH
OH
HO
OH
Maltulose
Lactulose
OH
[48]
[51]
OH
OH
HO
HO
OH
O
OH HO
O
O
HO
OH
HO
HO
OH
O
OH
OH
OH
OH
Nigerose
Cellobiose
[52]
[45]
HO OH
O
HO
OH
HO
HO
OH
O
Raffinose
HO
OH
O
OR
HO
OH
HO
6-O-Tosyl-glucose derivatives
OH
OH
R = H, Me(), allyl ()
[53]
[54]
OH
O
HO
HO
OH
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
OH
OH
OH
D-Mannitol
D-Sorbitol
[48, 55]
OH
D-Maltitol
[48]
[48, 55]
OH
OH
HO
HO
OH
OH
O
OH
HO
HO
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
-D-Glucopyranosyl-(1,6)- D-sorbitol
[48]
[48]
Fig. 2 (Continued)
OH
-D-Glucopyranosyl-(1,6)- D-mannitol
OH
OH
OH
HO
HO
HO O
HO
OH
OH
OH
HO
OH
OH
OH
Trehalulose
-D-arabino-Hexos-3-ulopyranosyl-(1,6)- D-fructose
OH
[48]
[48]
OH
OH
HO
HO
O
OH
HO
HO
OH
OH
OH
O
OH
OK
OH
OH
-D-arabino-hexos-3-ulopyranosyl-(1,6)-D-mannitol
[55]
HO
OH
OH
-D-Glucopyranosyl-(1,6)-D-arabinonic acid
[48]
HO
CH 2 OH
HO
HO
HO
OH
O
OH
O
O
OH
OH
OH
-D-Fructofuranosyl- -D-fructofuranosyl-(1,2':2,3')-dianhydride
Salicin
[246]
[48]
OH
HO
HO
OH
HN
O
OH
HO
OH
O
O
HO
OH
OH
O
O
HO
Acarbose
OH
OH
OH
[56]
HO
HO
OH
OH
O
HO
OH
O
OH
OH
OH
OCH 3
BocHN
O
OCH 3
BocHN
OH
OH
BocHN
[57]
[57]
HO
HO
D-glucal
[48, 55]
Fig. 2 (Continued)
Cl
(CH 2 )
OH
H 3C
(CH 2 )
n=2, 4, 6
OH
n
n=0, 1, 2, 3, 4...
Primary alcohols
(methanol, ethanol, propanol, butanol...)
Chloro derivatives
(2-chloroethanol, 4-chlorobutanol, 6-chlorohexanol)
[57]
[57, 58]
OH
OH
OH
HO
HO
OH
OH
OH
OH
OH
OH
Myricetin
Quercetin
[59, 60]
[60]
OH
OH
OH
HO
HO
OH
OH
OH
OH
OH
Luteolin
Ampelopsin
[60]
[61]
OH
OH
OH
HO
OH
OH
OH
O
O
HO
OH
O
OH
OH
HO
OH
O
Epigallocatechin gallate
Astragalin
[62]
OH
[63]
HO
HO
OH
COOH
HO
NH2
OH
HO
Phenol
Salicyl alcohol
[64]
[64]
OH
L-DOPA
OH
Catechol
[65]
[66]
HO
OH
OH
H
O
HO
OH
OH
OCH 3
3-Methoxycatechol
[66]
H 3C
CH 3
4-Methylcatechol
3-Methylcatechol
[66]
OH
[66]
Fig. 2 (Continued)
HO
L-ascorbic acid
[67]
OH
OH
OH
HO
H 3C
HO
HO
OH
OH
HO
OH
O
HO
OH
HO
NHAc
N-Acetyl-D-glucosamine
OH
[41]
[41]
[68]
OH
HO
O
HO
HO
HO
HO
OH
OH
Maltose
L-Rhamnose
OH
O
HO
OH
OH
OH
Arbutin
Salicin
[69]
[70, 71]
OH
OH
OH
HO
HO
HO
HO
O
OH
OH
HO
Piceid
Aesculin
OH
[71]
[72]
HO
HO
O
OH
HO
Hydroquinone
Caffeic acid
[72]
HO
OH
OCH 3
Vanillin
OH
[71]
Zingerose
[71]
[71]
OH
HO
OH
OH
OH
O
HO
O
HO
OH
OH
OH
(+) Catechin
D-Arabinose
[73]
OH
OH
OH
OH
OH
O
HO
HO
HO
OH
OH
OH
[74]
[74]
HO
OH
OH
OH
L-Arabinose
[74]
OH
OH OH
OH
D-Allose
OH
O
OH
HO
[74]
[74]
HO O
HO
OH
D-Xylose
[74]
O
OH
OH
D-Altrose
OH
OH
HO
HO
OH
D-Mannose
OH
[74]
OH
OH
O
HO
OH
D-Galactose
HO
D-Fucose
[74]
OH
OH
L-Fucose
[74]
L-Galactose
[74]
HO
HO
OH
O
OH
OH
OH OH
HO
L-Mannose
OH
OH
OH
OH
L-Xylose
L-Altrose
[74]
OH
OH
O
HO
OH
OH
OH
[74]
HO
OH
O
L-Allose
[74]
[74]
OH
OH
OH
OH
OH
HO
OH
HO
OH
OH
HO
OH
OH
OH
D-Sorbitol
OH
OH
D-Arabitol
D-Mannitol
[74]
[74]
[74]
OH
O
HO
HO
OH
OH
O
OH
HO
OH
OH
HO
HO OH
HO
OH
OH
HO
OH
OH
D-Xylitol
[74]
OH
OH
D-Maltitol
[74]
Myo-inositol
[74]
Fig. 3 (Continued)
1 N
746
Domain V
Domain N
1751
90
Domain B
184
793
1639
Domain IV
260
Domain B
927
1605
990
1591
Domain B
395
460
Domain A
550
Domain C
Domain A
1238
636
1377
Domain C
B
GH13 amylosucrase from N. polysaccharea (PDB 1G5A)
R446
Subsite +1
D394
Subsite +1
D1136
R509
D393
Q1140
W1065
H1135
D507
N1411
H392
E328
F250
D144
R284
R1023
D1458
E1063
D1025
D286
Y1465
Y147
Subsite -1
H187
Subsite -1
D1504
Q1509
Fig. 4 Structural comparisons of glucansucrases from GH13 and GH70 families. (A)
Overall three-dimensional structure of amylosucrase from N. polysaccharea (pdb:1G5A)
and glucansucrase GTF180 from L. reuteri (pdb:3KLK). (B) Representation of subsites 1
and 1 constituting the bottom of active site pocket, docked with sucrose (extracted
from pdb:1JGI and pdb:3HZ3, respectively). Subsites of glucansucrases from both GH13
and GH70 families share a similar spatial organization and involve comparable hydrogen
bonding networks mainly due to subsite 1. Catalytic residues are highlighted in bold and
water molecules are repredented as spheres.
Along with these reports, combinatorial engineering have been attempted to increase the specific activity of glucansucrases. The performance of amylosucrase from Neisseria polysaccharea (NpAS) was
enhanced by random mutagenesis, gene shuing and selective screening. Variants with up to a five fold increase in activity toward sucrose were
isolated (R20C/F598S and V389L/N503I).83 Variants with increased polymerization eciency (E227G), thermostability (P157A/D231Y, P234L/
G554S and N387D) or activity (N76D, E62K/D506N, N387D, Q613H) were
further isolated.84 Random engineering strategies may thus be useful for
enhancing the performances of recombinant enzymes what is of prime
interest for biotechnological purposes. Moreover, industrial processes
often require high reaction temperatures and the enhancement of enzyme thermostability is still challenging. Directed evolution of NpAS by
error-prone PCR has been performed and led to the isolation of two
double mutants (R20C/A451T and A170V/Q353L) and a single mutant
(P351S) with 3.5 up to 10 fold increased half-lives at 50 1C as compared to
the parental wild-type enzyme. The increased stability was suspected to
be due to the introduction of additional hydrogen-bonding interactions
and salt-bridge rearrangements that are assumed to strengthen the
overall structure.85
used for the selection of sucrose-utilizing transglycosylases. E. coli competent cells unable to use sucrose, transformed by a plasmid containing
an engineered gene coding for an amylosucrase activity, were grown on
solid medium containing sucrose and bromothymol blue (BTB) as pH
colour indicator. Cells expressing an active amylosucrase variant were
able to use sucrose and synthesize an amylose-like polymer. The fructose
released during the polymerization reaction was metabolized by E. coli
cells through glycolysis pathway to synthesize acidic products, the local
acidification being detected by BTB color change from blue to yellow.118
Another method has been developed for isolating E. coli cells displaying
intracellular dextransucrase activities that can be identified through a
polymer-forming based strategy. E. coli transformants are grown on solid
medium supplemented by 2% of sucrose. Clones displaying dextransucrase activity synthesize an extracellular glucan and can thus be
detected.119 Recently, a powerful medium-throughput screening of glucansucrase specificity has been developed. Product specificities of more
than 4,000 glucansucrase variants generated by combinatorial engineering were screened through a quantitive and highly sensitive NMR
based-approach with a rate of 480 variants per day. Altogether, 303
variants were successfully identified for their altered specificity underlining the potential of this method in glycomics for screening natural
glucan biodiversity.103 Surface Plasmon Resonance spectroscopy has
also been used for the detection of transglycosylase-catalyzed polymer
synthesis and the determination of enzymatic activity using the alternansucrase from Leuconostoc mesenteroides NRRL B-1355. Such a methodology might be used for glucan-synthesizing enzyme screening.120
Prospects
Variant
Sequence
Variant
WT
Sequence
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
R. Irague, S. Massou, C. Moulis, O. Saurel, A. Milon, P. Monsan, M. RemaudSimeon, J. C. Portais and G. Potocki-Veronese, Anal Chem, 2011, 83, 1202
1206.
R. Irague, A. Rolland-Sabate, L. Tarquis, J. L. Doublier, C. Moulis, P.
Monsan, M. Remaud-Simeon, G. Potocki-Veronese and A. Buleon, Biomacromolecules, 2012, 13, 187195.
S. Kralj, W. Eeuwema, T. H. Eckhardt and L. Dijkhuizen, Febs J, 2006, 273,
37353742.
S. Kralj, I. G. van Geel-Schutten, E. J. Faber, M. J. van der Maarel and
L. Dijkhuizen, Biochemistry, 2005, 44, 92069216.
S. S. van Leeuwen, S. Kralj, W. Eeuwema, G. J. Gerwig, L. Dijkhuizen and
J. P. Kamerling, Biomacromolecules, 2009, 10, 580588.
S. S. van Leeuwen, S. Kralj, G. J. Gerwig, L. Dijkhuizen and J. P. Kamerling,
Biomacromolecules, 2008, 9, 22512258.
H. Hellmuth, S. Wittrock, S. Kralj, L. Dijkhuizen, B. Hofer and J. Seibel,
Biochemistry, 2008, 47, 66786684.
H. K. Kang, A. Kimura and D. Kim, J Agr Food Chem, 2011, 59, 41484155.
S. Kralj, S. S. van Leeuwen, V. Valk, W. Eeuwema, J. P. Kamerling and
L. Dijkhuizen, Febs J, 2008, 275, 60026010.
J. Schneider, C. Fricke, H. Overwin, B. Hofmann and B. Hofer, Appl Environ
Microbiol, 2009, 75, 74537460.
, C. Moulis, J. Boutet, K. Descroix, S. Morel,
E. Champion, I. Andre
P. Monsan, L. A. Mulard and M. Remaud-Simeon, J Am Chem Soc, 2009, 131,
73797389.
, E. Champion, C. Moulis, S. Morel, P. Monsan,
L. A. Mulard, I. Andre
M. Remaud-Simeon and J. Boutet, European Patent Apllication, 2008, EP 2
100 966 A101.
E. Champion, F. Guerin, C. Moulis, S. Barbe, T. H. Tran, S. Morel,
K. Descroix, P. Monsan, L. Mourey, L. A. Mulard, S. Tranier, M. Remaud, J Am Chem Soc, 2012, 134, 1867718688.
Simeon and I. Andre
S. Emond, P. Mondon, S. Pizzut-Serin, L. Douchy, F. Crozet, K. Bouayadi,
H. Kharrat, G. Potocki-Veronese, P. Monsan and M. Remaud-Simeon,
Protein Eng Des Sel, 2008, 21, 267274.
S. Emond, G. Potocki-Veronese, P. Mondon, K. Bouayadi, H. Kharrat,
P. Monsan and M. Remaud-Simeon, J Biomol Screen, 2007, 12, 715723.
E. Champion, C. Moulis, S. Morel, L. A. Mulard, P. Monsan, M. Remaud, ChemCatChem, 2010, 2, 969975.
Simeon and I. Andre
S. R. Lee, A. R. Yi, H. G. Lee, M. U. Jang, J. M. Park, N. S. Han and T. J. Kim,
J Microbiol, 2011, 49, 320323.
C. Cle, A. P. Gunning, K. Syson, L. Bowater, R. A. Field and S. Bornemann,
J Am Chem Soc, 2008, 130, 1523415235.
C. Jackel and D. Hilvert, Curr Opin Biotech, 2010, 21, 753759.
E. M. Brustad and F. H. Arnold, Curr Opin Chem Biol, 2011, 15, 201210.
N. Tokuriki and D. S. Tawfik, Science, 2009, 324, 203207.
H. M. Senn and W. Thiel, Curr Opin Chem Biol, 2007, 11, 182187.
H. M. Senn and W. Thiel, Angew Chem Int Ed Engl, 2009, 48, 11981229.
U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore and
K. Robins, Nature, 2012, 485, 185194.