Download Domain Swapped Library and Screening for Expanding Substrate

Expanding substrate specificity of GT-B fold
glycosyltransferase via domain swapping and high-throughput
screening
Supplementary material
Sung-Hee Park1, Hyung-Yeon Park2, Jae Kyung Sohng3, Hee Chan Lee3, Kwangkyoung Liou3,
Yeo Joon Yoon 4, Byung-Gee Kim1, 5,*
1
Institute of Molecular Biology and Genetics, Interdisciplinary Program for Bioengineering,
Seoul National University, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea,
2
Department of Chemistry, Inha University, Incheon 402-751, Korea,
3
Institute of Biomolecule Reconstruction, SunMoon University, Chungnam 336-708, Korea,
4
Division of Nano Sciences, Ewha Womans University, 11-1, Daehyun-dong, Seodaemun-gu,
Seoul 120-750, Korea,
5
Institute of Bio Engineering, School of Chemical and Biological Engineering, Seoul National
University, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea.
Phone) +82-2-880-6774, Fax) +82-2-876-8945, E-mail) [email protected]
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Protein Modeling of HMT31
Homology modeling technique allows us to predict 3-D structure of a protein and to
identify key residues surrounding the active site pocket. The primary requirement in
constructing structural model of HMT31 is to select a proper template structure from
sequence alignment. To identify suitable parent standard structure for docking, we used the
FUGUE (Shi et al. 2001) sequence-structure homology recognition program. The alignments
produced by FUGUE for the highest-scoring hits were formatted with JOY (Mizuguchi et al.
1998) and analyzed visually to highlight the conservation of structurally important residues.
Profile-profile matching between the target sequence and the HOMSTRAD database
(Montalvao et al. 2005) was conducted for homology recognition and alignments. The
highest-scoring hit was glycogen synthase (AGt) (PDB codes = 1rzu) (Buschiazzo et al. 2004)
and Z-score was 4.24 (ID % = 16.2). The model was constructed based on the 1rzu with
ORCHESTRA (Schrauber et al. 1993) based on the result of FUGUE. The determined model
structure was validated by PROTABLE (Mannhold et al. 1995) and visual inspection was
performed in parallel by using 3-D graphics software. The initial model was energyminimized by the conjugate gradient method until energy gradient norm converged to 0.01
kcal/mol. Minimized enzyme structure and its binding site identified by SITEID (SYBYL,
Tripos Inc.).
Catalytic key residues and linker sequences of HMT31
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The linker loop sequence and catalytic key residues for domain swapping of the GT-B fold
enzyme were determined via protein modeling and ligand docking. The secondary structurebased alignment between AGT (PDB codes = 1rzu) (Buschiazzo et al. 2004) and HMT31 was
performed to predict the secondary- and tertiary structure of HMT31. The structure of
HMT31 belonged to the typical GT-B (Coutinho et al. 2003) fold. The modeling result
showed
that
the
linker
sequence
210FVGRIAHEKGWRHANNQSAYRRYGEPLNSRR240.
of
The
HMT31
was
210FVGRIAHEKGWRHA223
and 224NNQSAYRRYGEPLNSRR240 sequences were from KanF and GtfE, respectively. The
ligand docking study showed that Asp35, Asn38, Arg141, His374, Gly378 and Gln399 were
key catalytic residue of HMT31.
Multiple alignments result of HMT31 and Gtfs (GtfA, B, C, D and E) which were well
known GT-B fold enzymes involved in vancomycin related antibiotics is shown in the Figure
S9. The Asp35 of HMT31 was matched with the Asp13 as a catalytic general base in GtfA
(Mulichak et al. 2003), which is the GT in chloroeremomycin biosynthetic pathway. The
Arg141 of HMT31 was conserved in the hyper-variable loop region (Mulichak et al. 2001).
The loop region participates in the formation of an acceptor binding pocket like that observed
in the N-terminal domains of Gtfs. His374 and Gly378 constructed the “HHXXAGT” loop
(Hu and Walker 2002), which is a highly conserved Gly-rich sequence and a representative
binding site of -phosphate of the NDP-sugar donor in GtfA (Mulichak et al. 2003). The
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Gln399 is located in the loop region of the // motif (Hu and Walker 2002), constituting a
large part of the donor binding site in the GT-B fold structure, which comprises GtfB
(Mulichak et al. 2001), GtfA (Mulichak et al. 2003) and MurG (Hu et al. 2003).
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Color Assay Procedure.
Figure S1. Schematic representation of color assay screening method for
glycosyltransferase. (a) Bacterial colonies expressing glycosyltransferase on a LB plate
containing IPTG (0.01 mM) and cresol red (0.05 mM), (b) Bacterial colonies on a replica
filter paper, (c) Incubation of the replica filter paper in a cresol red dipping solution
containing acceptor and donor substrate, (d) Color change observation of active
glycosylated colonies (Yellow).
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Library construction.
Figure S2. Lane 1: Size marker; Lane 2: Parental vector digested with HindIII; 3: Size
distribution of library after ExoIII and Mungbean nuclease treatment.
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Whole-cell color change.
Figure S3. pH color assay result for bacterial streaks. (upper) Yellow color of the KE
chimera harboring E. coli BL21, (lower) Red color of the negative control, pET28a
vector harboring E. coli BL21.
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MS analysis of mono-glucosylated 2-DOS.
Figure S4. ESI-MS spectrum for the glucosylation of 2-DOS catalyzed by HMT31. 2deoxystreptamine (2-DOS) is found at m/z 163.15 [M + H]+ and mono-glucosylated 2DOS is found at m/z 325.00 [M + H]+ and at 347.04 [M + Na]+ respectively.
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Figure S5. (a) Calibration curve: relationship between proton concentration and
absorbance at 436 nm. (b) Reaction velocity of HMT31. The kinetic parameters for the
HMT31 were determined on varied acceptor as 2-deoxystreptamine (2-DOS) and fixed
donor 10mM dTDP-glucose.
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Figure S6. Spectrometric data of HMT31 reaction toward NDP-sugars and 2-DOS,
based on pH sensitive assay.
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Figure S7. Sequence alignment of target #31 and the hit template AGT (PDB code =
1RZU) chosen from FUGUE. -helix and β-sheet are labeled a and b, respectively.
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Figure S8. Structure and active pocket of HMT31. (a) A ribbon representation of
HMT31 highlights the Rossmann-like fold in each domain as well as the location of TDP
binding. (b) Schematic representation showing the interaction between HMT31 residues
and TDP. Observed hydrogen bonding and ionic interactions are depicted with dashed
lines indicating the distances.
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Figure S9. Multiple alignment result of #31 and Gtfs. GtfA, GtfB and GtfC:
chloroeremomycin biosynthetic pathway; GtfD and GtfE: vancomycin pathway.
S13
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