3D structure of Geobacillus GtfC: GH13 or GH70? Background & goal

Abstract

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes are of interest for modification of starch into low-glycemic index food ingredients [1]. GtfCs have been classified in Glyco-side Hydrolase family 70 (GH70), together with glucansucrases and GtfB-type α-GTs from lactic acid bacteria (LAB). However, GtfCs have been identified only in non-lactic acid bacteria and do not show circular permutation of the catalytic domain. Therefore, GtfCs have been proposed as evolutionary intermediates between starch-degrading α-amylases from Glycoside Hydrolase family 13 (GH13) and starch-converting GtfB enzymes from GH70 [2], but 3D structures have not been resolved.
We report the first crystal structure of a GtfC, GbGtfC-ΔC from Geobacillus 12AMOR1, revealing high structural similarity with most GtfBs, but lacking circular permutation. We propose that GtfC enzymes evolved from hydrolytic GH13 α-amylases by acquiring transglycosylation capabilities and a single-segment auxiliary domain IV, before the events that resulted in permutation of the catalytic domain A. The GbGtfC structure likely repre-sents that of other GtfCs, although it has a so far unique alternating α-1,4/α-1,6 product specificity [3] determined by non-conserved residues near acceptor binding subsites +1/+2.

Full text

3D structure of Geobacillus GtfC:
GH13 or GH70?
Tjaard Pijning1, Evelien te Poele2, Tijn de Leeuw2, Albert Guskov1and Lubbert Dijkhuizen2
1Biomolecular X-ray Crystallography, Groningen Biomolecular Sciences and Biotechnology Institute, Unive rsity of Groningen, Nijenborgh 7, 9747 AG Groningen,The Netherlands
2 CarbExplore Research BV, Zernikepark 12, 9747 AN Groningen, Groningen, The Netherlands
Background &goal
GtfC-type 4,6-α-glucanotransferases (α-GTs) are
of interest for modification of starch into low-
glycemic food ingredients1.They have been
classified in Glycoside Hydrolase family 70
(GH70), together with glucansucrases and GtfB-
type α-GTs from lactic acid bacte ria (LAB).
However, GtfC-type α-GTs:
▪occur in non-LAB
▪share low sequence identity
▪lack circular permutation
GtfCs therefore have been proposed as evo-
lutionary intermediates between starch-
degrading GH13 α-amylases and α-glucan
synthesizing GH70 GtfBs2.
We determined the first crystal structure of a
GtfC, GbGtfC-ΔCfrom Geobacillus 12AMOR13,
compared its 3D structure, and performed a
phylogenetic and structural analysis of 63
putative GtfC-type α-GTs.
Structure determination
AHis-tagged and C-terminally truncated con-
struct of Geobacillus 12AMOR1GtfC (GbGtfC-
ΔC, residues 33-738)2was crystallized by vapor
diffusion at 293 Kusing 1.07-1.14 M(NH4)2SO4,
0.1 M MES-NaOH, pH 6.5, 0.4 M Na3Citrate.
The GbGtfC-ΔCcrystal s tructure was deter-
mined by molecular replacement.
Phylogenetic analysis of GtfCs
An NR-BLASTp search with the Geob acillus
12AMOR1sequence was performed;only hits
showing acomplete catalytic domain (pre sence
of GH70 homology motifs I-IV) and circular
permutation were selected.
Aphylogenetic tree was constructed including
other GH70 enzymes (glucansuc rases, GtfBs,
GtfDs) as well as GH13_5 α-amylases.Before
alignment, permuted sequences were first
‘depermuted’.
Comparison with GtfB-type α-GTs
t.pijning@rug.nl
Domain IV of GbGtfC is a110-
residue single segment insertion
in domain B, partially overlap-
ping with that of the dual
segment domain IV of the GtfB-
type 4,6-α-GT from Lactobacillus
reuteri 1214(shown in grey).
Conclusions &Outlook
We determined the first crystal structure of a
GtfC-type 4,6-α-glucanotransferase (GbGtfC-
ΔC).Its core doma in topology and ac tive site
architecture is highly conserved with most
GtfB-type 4,6-α-GTs found in LAB, despite low
sequence similarity, despite the absence of
circular permutation in the catalytic domain,
and despite the fact that GtfC-(and G tfD-)type
starch-converting enzymes evolved in non-LAB.
GbGtfC represents the so far 63 putative GtfCs
found in databases, with atunneled
architecture of the binding groove.We thus
expect that GtfC-type 4,6-α-GTs synthesize
linear α-glucans.The so far unique specificity of
GbGtfC, leading to alternating α-1,4/α-1,6
linkages in its produc ts, is currently under
investigation through mutation and docking
studies.
1.Gangoiti,J. et al., Cr it.Rev.Food Sci.Nutr. (2020), 60,123 -146.2.Gangoiti, J. et al., Appl.Environm.Microbiol. (2015), 82,756-766.3.Te Poele, E.M. et al., J. Agric.Food Chem. (2021), 69,9859-9868.4.Bai, Y. et al., Struc ture
(2017), 25,231-242.5.Pijning, T. et al., J. Agric.food Chem. (2021), 69,13235-13245.6.Jumper et al.,Nature (2021), 596,583-589.
Parameter
Resolution (Å)
Rpim
Unique observations
Multiplicity
I/σ
Completeness (%)
CC1/2
overall
131.3-2.25
0.030
59408
1.9
20.5
99.3
0.999
highest shell
2.31-2.25
0.245
4359
1.9
4.2
95.0
0.795
V 
IVn
761-898
IVc
1581-1614
IV
223-332
Loop
A1
Loop B
Loop
A2
D517
D413
E446
GbGtfC crystal structure
The GbGtfC-ΔCcrystal structure showing the
canonical GH70 domain arrangement but with a
non-permuted ca talytic domain A. The
GH13/GH70 core consists of domains A +B+C;
the auxiliary doma in IV is a110-residue single-
segment insertion in domain B.
The catalytic triad of GbGtfC contains D413 (nucleophile), E446
(acid/base) and D517 (transition state stabilizer).Three loops near
the active site (loops A1, A2 and B) create atunneled binding
groove in GbGtfC, similar to L. reuteri 121 GtfB4,5. A sta rch fragment
(maltooctaose) was modeled in the binding groove based on crystal
structures of other GH70 4,6-α-GTs and GH13 α-amylases.
Modeling other GtfC-type α-GTs
+3 +2 +1 -1 -2
-3
-4
-5
Heyndrickxia
sporothermodurans
seq id =76.3%
pLDDT = 94.9
Geobacillus
12AMOR1
pLDDT = 94.8
Exiguobacterium
sibiricum
seq id = 56.3%
pLDDT = 94.6
Exiguobacterium
acetylicum
seq id = 56.5%
pLDDT = 94.3
Weizmannia
coagulans
seq id = 72.6%
pLDDT = 94.8
Ig2a
Ig2b
Ig2a
Ig2b
Ig2a
Ig2b
Ig2a
Ig2b
SH3a
SH3b
SH3c
AlphaFold6models of
selected GtfC-type α-GTs;
N-terminal residues with
pLDDT <60 are not shown.
C-terminal domains have
Bacterial Immunoglobulin
type 2(Ig2)-like or SRC
Homology 3(SH3) folds.
Superposition of the active sites with the loops
A1, A2 and Bshow that each of these GtfCs
feature atunneled binding groove like GbGtfC.
Phylogenetic analysis
Unrooted phylogenetic tree and proposed evolutionary pathways for GH70 and GH13 sequences.GH13 α-
amylases (present in all kingdoms of life) acquired transglycosylation specificity by changing their active
site, and insertion of domain IV resulted in a(still non-permuted) GH70 ancestor α-GT.Fro m the ancestor,
two branches evolved:in non-LAB, GtfC-and GtfD-type α-GTs remained non-permuted and acquired C-
terminal Ig2- or SH3-domains.The second branch evolved in LAB:GS (glucansucrases) and BrS (branching
sucrases) became circularly permuted, acquiring different auxiliary domains (e.g. domain V).
GH13 GH70
Core
active site rearrangements;
domain IV insertion
gene duplication;
partial termini deletion;
insertion into
domain IV
non-permuted
α-amylases
non-permuted
ancestor
α-GT
(domain
insertion)
non-
LAB
LAB
non-permuted
GtfC/D-like
α-GT
permuted
GtfB-like
α-GT;
GS; BrS
Previous characterization of GbGtfC products3,4
showed that the enzyme synthesizes alinear α-
1,4/α-1,6 alternating α-glucan, while GtfB
products contain consecutive α-1,6 linkages.
G1
G2
G3
G4
G5
G6
G7
M |maltoheptaose| amy lose V
ctrl GtfC GtfB ctrl GtfB GtfC
GtfB product
GtfC product
R / Rfree:
0.252 / 0.292
RMSD (bonds/angles):
0.006 Å / 1.38o
Average B(pr otein):
55.0 Å2
IV
B
A
C
active
site
26
735
Superposition with
L. reuteri 121 GtfB-
ΔNΔV4(grey).