Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′-Deoxyribosyltransferase

Article abstract of DOI:10.1002/cbic.201900397

The use of nucleoside 2′-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2′-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDT_Y5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine?adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDT_Y5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.

Full text of DOI:10.1002/cbic.201900397

Accepted Article
Title: Structure-guided tuning of a selectivity switch towards
ribonucleosides in Trypanosoma brucei purine nucleoside 2'
deoxyribosyltransferase
Authors: Jon Del Arco, Alberto Mills, Federico Gago, and Jesús
Fernández-Lucas
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To be cited as: ChemBioChem 10.1002/cbic.201900397
Link to VoR: http://dx.doi.org/10.1002/cbic.201900397
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
Structure-guided tuning of a selectivity switch towards ribonu-
cleosides in Trypanosoma brucei purine nucleoside 2'-deoxy-
ribosyltransferase
Jon Del Arco,[a] Alberto Mills,[b] Federico Gago,[b] Jesús Fernández-Lucas*[a,c]
Abstract: The use of nucleoside 2’-deoxyribosyltransferases
(NDTs) as biocatalysts for the industrial synthesis of nucleoside
analogues is often hindered by their strict preference for 2’-
deoxyribonucleosides. We now show that a highly versatile purine
nucleoside 2’-deoxyribosyltransferase from Trypanosoma brucei
(TbPDT) can also accept ribonucleosides as substrates, most
likely because of the distinct role played by Asn53 at a position
that is usually occupied by Asp in other NDTs. Moreover, this
unusual activity was improved ~3-fold by introducing a single
amino acid replacement at position 5 following a structure-guided
approach. Biophysical and biochemical characterization revealed
that the TbPDTY5F variant is a homodimer that displays maximum
activity at 50 oC and pH 6.5 and shows a remarkably high melting
deoxyribosyltransferases (NDTs)[3] (EC 2.4.2.6) catalyze a
transglycosylation reaction consisting of the exchange of the
2′-deoxyribose moiety between a purine and/or pyrimidine
nucleoside and a purine and/or pyrimidine base.[4] NDTs are
highly specific for 2′-deoxyribonucleosides and generally
display poor activity on modified C2′ and C3′ nucleosides.[4b, 5]
The fact that no wild-type NDT or any of their reported variants
has shown activity on ribonucleosides so far is seen as an
undesirable substrate limitation. The present work describes,
for the first time to the best of our knowledge, the ability of a
versatile purine NDT (PDT) from Trypanosoma brucei
(TbPDT)[4b] to use ribonucleosides as substrates (Scheme 1)
and how this unusual ribosyltransferase activity was enhanced
in an engineered Y5F enzyme variant. We also demonstrate
that this decreased stringency towards the pentose relies
heavily on the presence of Asn53 in a position usually
occupied by Asp in other family members.
o
temperature of 69 C. Substrate specificity studies demonstrated
that 6-oxopurine ribonucleosides are the best donors (inosine >
guanosine >> adenosine) whereas no significant preferences exist
between 6-aminopurines and 6-oxopurines as base acceptors. In
contrast, no transferase activity could be detected on xanthine and
7-deaza purines. TbPDTY5F was successfully employed in the
synthesis of a wide range of modified ribonucleosides containing
different purine analogues.
The use of modified nucleosides as antitumor, antiviral and
antimicrobial drugs is well established.[1] Because of this,
interest in stereoselective, sustainable and cheap synthetic
methods keeps growing in the pharmaceutical industry. Due to
their versatility under mild reaction conditions, together with
unique chemo-, regio- and enantioselectivities, enzymatic
approaches are partially or totally replacing traditional multi-
step chemical methods, which often require the presence of
organic reagents and tedious purification processes. In this
context, glycosyltransferases arise as cost-effective and
sustainable alternatives for the enzymatic syntheses of a wide
variety of natural nucleosides and nucleoside analogues (NAs)
by employing direct one-pot transglycosylation reactions,[2]
most frequently those involving a purine and/or pyrimidine
nucleoside as the donor and a purine and/or pyrimidine
nucleobase as the acceptor. In this regard, nucleoside 2′-
Scheme 1. Purine nucleoside 2’-ribosyltransferase activity of TbPDT.
A
multiple sequence alignment of TbPDT versus
Leishmania mexicana PDT (LmPDT) and their closest fully
characterized homologous NDTs from Lactobacillus species
reveals that PDTs from trypanosomatides display the
canonical “NDT domain” (Pfam PF05014)[4a] that is also
shared by nucleoside and 2´-deoxynucleoside 5’-
monophosphate N-glycosidases. These latter enzymes
catalyse the hydrolytic cleavage of the glycosydic bond to yield
a free nucleobase and the corresponding phosphorylated
sugar.[6] Interestingly, the strict specificity of N-glycosidases
towards 2′-ribosyl or 2′-deoxyribosyl groups was attributed,
upon resolution of the X-ray crystal structure of Streptomyces
rimofaciens cytidine-5'-monophosphate (CMP) hydrolase
(SrMilB) in complex with CMP, to the presence of either a Phe
or Tyr side chain close to the catalytic Glu residue. Further
biochemical work indeed revealed that the replacement of Phe
with Tyr in SrMilB results in a dramatic reversal of substrate
specificity from CMP to dCMP.[6d]
[a]
Jon del Arco, Dr. J. Fernández-Lucas
Applied Biotechnology Group
Universidad Europea de Madrid
E-28670 Villaviciosa de Odón, Madrid (Spain)
E-mail: jesus.fernandez2@universidadeuropea.es
Alberto Mills, Prof. F. Gago
Department of Biomedical Sciences and “U. A. IQM-CSIC”
School of Medicine and Health Sciences
University of Alcalá, E-28805 Alcalá de Henares, Madrid (Spain)
Dr. J. Fernández-Lucas
[b]
[c]
Inspired by these reported findings and knowing the relative
laxity of wild-type TbPDT in the recognition of both C2′- and
C3′-modified nucleosides,[4b] we reasoned that the reverse
replacement of Tyr with Phe at the equivalent position in
Grupo de Investigación en Ciencias Naturales y Exactas, GICNEX,
Universidad de la Costa, CUC, Calle 58 # 55 – 66, Barranquilla,
Colombia.
1
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
TbPDT could enhance the ability of this enzyme to accept
ribonucleosides as substrates. Molecular dynamics (MD)
simulations of wild-type and variant enzymes revealed
hydrogen-bonding patterns in the active site (Figure S3) that
made us think that this unusual tolerance to the C2’ hydroxyl
is likely due to the presence of Asn53 in a position generally
occupied by an aspartic acid in other members of the family[3-
4] (Figure 1). Thus, to assess the role of both Tyr5 and Asn53
in sugar recognition and the distinct interplay among the sugar
hydroxyls and the side chains of residues Glu82, Asn53 and
Tyr5, the mutant genes ndtY5F, ndtN53D and ndtY5F/N53D were
cloned and overexpressed in E. coli BL21(DE3), as described
before, and the recombinant N-terminal His6-tagged variants
were purified by using Ni-affinity and size-exclusion
chromatography.[4b] SDS-PAGE analysis of the purified
enzymes showed only one protein band with an apparent
molecular mass of 19.0 kDa, which is very close to the value
calculated from their amino acid sequence (Figure S2).
(Table 1). Thus, the discrimination between 2’-deoxyribo- and
ribonucleosides was reduced by a factor of 10. Interestingly,
our MD simulation results showed that the single replacement
of Tyr by Phe at position 5 leads to an improved hydrogen-
bonding interaction between the 2′-OH from the ribose moiety
and one of the Glu82 carboxylate oxygens (OE1), so that the
other carboxylate oxygen (OE2) more easily adopts a proper
orientation for attack on the anomeric C1′ of the ribose (Figure
1). We note, however, that while this indicates feasibility of the
Michaelis complex, it does not inform about possible changes
in the energy barriers separating substrates and products
along the coordinates of the two half-reactions.
Table 1. 2’-deoxyribosyl- (dR) and ribosyltransferase (R) activities of wild-
type and variant TbPDTs using either deoxyinosine (dIno) or inosine (Ino)
as the nucleoside donor and adenine (Ade) as the base acceptor.
Specific activity
(IU/mg protein)
dIno
+ Ade[a]
Ino
+ Ade[b]
dR/R
selectivity ratio
pH 6.0c
Wild type
Y5F
38.5 ± 3.3
11.3 ± 2.2
11.0 ± 2.3
7.3 ± 1.2
0.9 ± 0.1
2.7 ± 0.3
0.2 ± 0.0
1.6 ± 0.0
42.8
4.2
55.0
4.6
N53D
Y5F/N53D
pH 4.0d
Wild type
Y5F
N53D
Y5F/N53D
47.5 ± 1.3
12.3 ± 2.4
47.5 ± 1.0
11.1 ± 1.5
<0.1 ± 0.0
0.5 ± 0.3
n.d.
475
24.6
47.5
11.1
<0.1 ± 0.1
[a] 0.33 μg of enzyme in 40 μL at 50 oC, 5 min. [Substrates] = 10 mM.
[b] 1 μg of enzyme in 40 μL at 50 oC, 5 min. [Substrates] = 2 mM.
[c] 50 mM sodium phosphate buffer.
[d] 50 mM sodium citrate buffer.
The biochemical characterization confirmed that TbPDTY5F
is, like TbPDTwt, a homodimer and revealed that it is active in
a 40-60 oC temperature interval (>60 % retained activity), with
o
a peak value at 50 C (Figure 2A). Moreover, the pH profile
showed >60 % retained activity in the pH range 5-7 and
optimal activity at pH 6-7 (Figure 2B). In view of these findings,
o
50 mM sodium phosphate pH 6.5 and 50 C were chosen as
suitable conditions for further studies. The thermal
denaturation curves[7] allowed us to estimate for TbPDTY5F
a
Tm of 69 oC (Figure 2C), which compares favorably with those
of NDTs from the mesophilic Lactobacillus reuteri (Tm = 64
oC)[5a] and Bacillus psychrosaccharolyticus (Tm = 49 oC).[8]
This unusually high Tm value is most likely due to the
presence of a disulfide bridge between the spatially very close
Cys77 and Cys77# residues from each monomer, as
suggested earlier for TbPDTwt.[4b] This hypothesis is supported
Figure 1. A) Schematic drawing of the binding of Ino within the active site of
wild-type TbPDT (left) and TbPDTY5F (right). The encircled areas focus on
the hydrogen-bonding side chains involved in orienting the carboxylate for
nucleophilic attack on C1’ (magenta arrow). B) 3D view of the active site of
TbPDTY5F (C atoms in green for one monomer and cyan for the other) in
complex with Ino (C atoms in grey). Enzyme residues relevant for the
discussion are shown as sticks and have been labeled. Yellow dashed lines
represent hydrogen bonds whereas the magenta broken line stands for the
direction of nucleophilic attack.
by the fact that an engineered C77S variant (TbPDTC77S
)
displayed, unlike TbPDTwt, a drastic loss of activity over the
course of 2 h upon incubation at 50 ºC for 8 h (Figure S3).
The hydrogen-bonding interaction network depicted in
Figure 1 is facilitated by the presence of Asn53 in a position
that is occupied by an aspartic acid in many other members of
the NDT family. The finding that this asparagine is strictly
conserved in trypanosomatidae[4] led us to assess the
interplay between the catalytic Glu82 and the spatially close
Tyr5 and Asn53 residues. The N53D substitution resulted in
Wild-type TbPDT (TbPDTwt) and its TbPDTY5F, TbPDTN53D
,
and TbPDTY5F/N53D variants were assayed for the enzymatic
synthesis of Ado and dAdo using Ino or dIno as the nucleoside
donors, respectively, and Ade as the base acceptor. The
single amino acid replacement Y5F resulted in a ~4-fold
decrease in 2’-deoxyribosyltransferase activity and a 3-fold
increase in ribosyltransferase activity depending on the pH
2
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
Figure 2. Ribosyltransferase activity and stability profiles of TbPDTY5F. A) Temperature dependence of TbPDTY5F activity. B) TbPDTY5F activity
in (○) 50 mM sodium citrate (pH 4-6), (●) 50 mM sodium phosphate (pH 6-8), and (∆) 50 mM sodium borate (pH 8-10). C) Melting of TbPDTY5F
as monitored by differential scanning fluorimetry.[7]
The biochemical characterization of TbPDTY5F was completed
a loss of activity at pH 6 comparable to that previously found for
TbPDTY5F whereas the activity at pH 4 was the same as in
TbPDTwt (Table 1). Remarkably, catalysis by the TbPDTY5F/N53D
variant containing both amino acid substitutions was as pH-
dependent as that of TbPDTwt.
by assessing its potential to use different natural purine
nucleosides and purine bases as donors and acceptors,
respectively (Table 2).
Table 2. Substrate specificity of TbPDTY5F
.
A dependency on pH for NDTs in general and TbPDT in
particular can be expected from the fact that attack of the catalytic
glutamate on C1’ is accompanied by proton transfer to N7 of the
purine ring from a carboxylic acid, which in the case of TbPDT
(Figure 1A) belongs to the side chain of Glu119 in the other subunit
making up the dimer (Figure 1B).[4b] Besides, the significant loss
of activity at pH 6 observed for TbPDTN53D (Figure 3) highlights a
significant evolutionary advantage of the wild-type enzyme
brought about by use of a protonation-independent asparagine
versus a protonation-dependent aspartic acid as the hydrogen
bond donor for purine N3 recognition. Poisson Boltzmann
electrostatic calculations[9] on TbPDTN53D provide an estimated
pKa value for Asp53 of 5.2 in both monomers in the absence of
substrate. Taken together, our results attest to the importance of
subtle hydrogen-bonding rearrangements in active site residues
to tip the balance towards catalysis or the stabilization of a non-
productive complex (Figure S1). These results are in accordance
TbPDTY5F
Acceptor
Ade
Gua
Hyp
Xan
Donor
Ado[a]
-
0.1 ± 0.0 0.1 ± 0.0
n.d.
Guo[b]
Ino[b]
2.3 ± 0.1
-
1.9 ± 0.1
-
n.d.
n.d.
2.7 ± 0.1 1.8 ± 0.1
[a] Reaction conditions: 1 μg of enzyme in 40 μL at 50 oC, 10 min.
[Substrates] = 1 mM, 50 mM sodium phosphate buffer, pH 6.5.
Reaction conditions: 1 μg of enzyme in 40 μL at 50 oC, 5 min.
[Substrates] = 2 mM, 50 mM sodium phosphate buffer, pH 6.5. n.d. =
not detected.
[b]
It is noteworthy that TbPDTY5F shows a remarkable preference
for ribonucleosides containing 6-oxopurine over those possessing
a 6-amino group; thus, the specific activities were 18-19 times
higher when Ino or Guo were used as donors relative to Ado. In
contrast, the nature of the base acceptor (Ade, Hyp and Gua) has
a negligible effect on the catalytic efficiency save for Xan, which
is inactive. In view of our MD simulation results, this finding is
most likely due to electrostatic repulsion between the 2-oxo group
of the base and carbonyl groups from the flexible recognition loop
encompassing 39TDNEATEA46 that connects β2 and α3
secondary structure elements (see Pro38 and Thr39 in Figure
1B).
with
previous
reports
for
NDTs
from
Bacillus
psychrosaccharolyticus[8] and Lactobacillus delbrueckii[10]
describing the interaction between the side chains of the catalytic
Glu and a positionally equivalent Tyr residue as essential for
catalysis.
Finally, to study the potential of TbPDTY5F as a biocatalyst, the
enzymatic synthesis of several ribonucleosides was carried out
using Guo as the donor and different substituted purines (2-ClAde,
2-FAde, 2,6-DAP, 6-ClPur, 6-MP, 6-MeOGua, 6-MetPur, 7-
deazaHyp, and 7-deazaXan) or benzimidazole (Bzm) as the
acceptors (Table 3). As was shown to be the case with
TbPDTwt,[4b] TbPDTY5F is highly tolerant to a broad range of purine
modifications but 7-deazapurine derivatives are not accepted as
substrates. This finding is in consonance with the proposed
Figure 3. Effect of pH on 2’-deoxyribosyltransferase activity in (A) TbPDTwt and
(B) TbPDTN53D. (∆) 50 mM sodium citrate (pH 4-6), (●) 50 mM sodium phosphate
(pH 6-8), and (○) 50 mM sodium borate (pH 8-10).
3
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
catalytic mechanism, which involves glycosydic bond cleavage by
nucleophilic attack of Glu82 on C1′ accompanied by proton
transfer from Glu119# to N7 of the purine ring (Figure 1B).[4b]
Experimental Section
Thermal denaturation
The melting temperature (Tm) was measured using differential
scanning fluorimetry in a CFX96 Real-Time PCR Detection
System (Bio-Rad, Spain) essentially as described.[7] Briefly,
TbPDTY5F was diluted to a final concentration of 20 µM and 18
µL were mixed with 2 µL 100x diluted SYPRO Orange (Sigma-
Aldrich, St. Louis, MO). The samples were heated from 35 to 95
°C at 1 °C/min. The fluorescent signal was measured with
excitation and emission filters of 500 and 580 nm, respectively.
The Tm was approximated to the melt peak obtained from a graph
plotting the negative first derivative of the melting curve with
respect to the temperature (dRFU/dT vs T), where RFU stands for
relative fluorescence units.
To avoid the reverse reaction that could be detrimental to overall
yields, the effect on enzyme activity of molar ratio (1 vs 3 mM
Guo) was also studied. In all cases, an excess of Guo led to
improved conversion rates (Table 3). Thus, it seems that an
excess of ribonucleoside partially shifts the reaction equilibrium
towards product formation. Consequently, these experimental
results support 3:1 as the optimal nucleoside:base molar ratio for
biotransformation using TbPDTY5F
.
Table 3. Synthesis of ribonucleoside analogues by TbPDTY5F using
guanosine as the ribosyl donor and several purine base derivatives or
analogues as the acceptors.
Donor
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Guo
Acceptor
Product
Molar
ratio
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
1:1
3:1
Conversion
(%) a
Computational Methods
Ade
Ado
35 ± 4
50 ± 2
35 ± 4
55 ± 1
n.d.
The Protein Data Bank entry 2A0K provided the Cartesian
coordinates for a 3D model of the apo form of TbPDT solved by
X-ray crystallography.[11] Replacement of Tyr5 by a Phe residue in
both subunits was accomplished by means of the interactive
molecular graphics program PyMOL,[12] which was also employed
for structure visualization, molecular editing and figure
preparation. Ado and Ino were docked into the active site of
TbPDTY5F as reported earlier for dAdo in LmPDT[4a] and dIno in
TbPDT.[4b] Each complex of TbPDTY5F with either Ino or dIno
bound in the active site of both monomers was immersed in a box
of ~ 13,400 TIP3P water molecules that extended 25 Å away from
any solute atom and 4 Na+ ions were added to ensure electrical
neutrality. Energy minimization followed by unrestrained
molecular dynamics simulations for 150 ns were carried out as
described before[4b] using the pmemd_cuda.SPFP module and the
standard ff14SB force field parameter set in AMBER14.[13] The
cpptraj module[14] in AMBER14 was employed for data processing
and geometry analysis of the calculated trajectories.
Hyp
Ino
Xan
Xao
n.d.
2-ClAde
2-FAde
2,6-DAP
6-ClPur
6-MP
2-ClAdo
2-FAdo
37 ± 1
69 ± 2
48 ± 2
68 ± 2
60 ± 2
67 ± 3
18 ± 1
24 ± 4
14 ± 3
50 ± 2
65 ± 4
83 ± 2
29 ± 4
41 ± 2
n.d.
2,6-DAPRib
6-ClPurRib
6-MPRib
6-MeOGuo
6-MetPurRib
7-deazaIno
7-deazaXao
BzmdRib
6-MeOGua
6-MetPur
7-deazaHyp
7-deazaXan
Bzm
ABBREVIATIONS
Ade, Adenine; Gua, Guanine; Hyp, Hypoxanthine; Xan, xanthine,
2-ClAde, 2-chloroadenine; 2-FAde, 2-fluoroadenine; 2,6-DAP,
n.d.
n.d.
2,6-diaminopurine;
6-ClPur,
6-chloropurine;
6-MP,
6-
n.d
mercaptopurine; 6-MeGua, 6-methoxyguanine; 6-MetPur, 6-
methylpurine; 7-deazaHyp, 7-deazahypoxanthine; 7-deazaXan,
7-deazaxanthine; Bzm, benzimidazole.
30 ± 3
35 ± 4
a Reaction conditions: 1 μg of enzyme in 40 μL at 50 oC, 30 min.
[Guo] = 1 or 3 mM, [Base] = 1 mM, 50 mM sodium phosphate buffer,
pH 6.5. n.d. = not detected.
dAdo, 2’-deoxyadenosine; dIno, 2’-deoxyinosine; Ado,
adenosine; Ino, inosine Guo, guanosine; 2-ClAdo, 2-
chloroadenosine; 2F-Ado, 2-fluoroadenosine; 2,6-DAPdRib, 2,6-
DAP riboside; 6-ClPurRib, 6-ClPur riboside; 6-MPRib, 6-MP
riboside; 6-MeOGuo, 6-methoxyguanosine, 6-MetPurRib, 6-
MetPur riboside; 7-deazaIno, 7-deazainosine, 7-deazaXao, 7-
deazaxanthosine and BzmRib, Bzm riboside.
In conclusion, Trypanosoma brucei purine nucleoside 2'-deoxy-
ribosyltransferase (TbPDT) is shown to be the first NDT that
accepts
ribonucleosides
as
substrates
and
the
deoxyribose/ribose selectivity ratio improves ~10 fold when Tyr5
is replaced with a phenylalanine using site-directed mutagenesis.
Biophysical and biochemical characterization revealed that the
TbPDTY5F variant is a homodimer that displays maximum activity
at 50 oC and pH 6.5 and shows a remarkably high melting
temperature of 69 oC. Substrate specificity studies demonstrated
that 6-oxopurine ribonucleosides are the best donors (inosine >
guanosine >> adenosine) whereas no significant preferences
exist between 6-aminopurines and 6-oxopurines as base
acceptors. In contrast, no transferase activity could be detected
on xanthine and 7-deaza purines. TbPDTY5F was successfully
employed in the synthesis of a wide range of modified
ribonucleosides containing different purine analogues.
Acknowledgements
This work was supported by grants XSAN001906 from the
Santander Foundation (to J.F.L.) and SAF2015-64629-C2-2-R
from the Spanish MEC/MICINN (to F.G.).
Keywords: biocatalysis • nucleoside analogues • 2‘-deoxy-
ribosyltransferase • enzyme immobilization • molecular dynamics
4
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
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10.1002/cbic.201900397
ChemBioChem
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Jon Del Arco,[a] Alberto Mills,[b] Federico
Gago,[b] Jesús Fernández-Lucas*[a,c]
Page No. – Page No.
Structure-guided tuning of a selectivity
switch towards ribonucleosides in
Trypanosoma brucei purine nucleoside
2'-deoxyribosyltransferase
Text for Table of Contents
A ~3 fold improvement in enzyme activity towards ribonucleosides of a purine 2'-deoxyribosyltransferase from Trypanosoma brucei
(TbPDT) was achieved upon replacement of Tyr with Phe at position 5 following a structure-guided approach. The TbPDTY5F variant
was biophysically and biochemically characterized and successfully employed in the synthesis of a wide range of modified
ribonucleosides containing different purine analogues.
6
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