(Received for publication, February 4, 1997, and in revised form, March 19, 1997)
From the Institute for Biological Sciences, National
Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6 and the
¶ Department of Chemistry, University of Alberta, Edmonton,
Alberta, Canada T6G 2G2
The human blood group A and B glycosyltransferase
enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 Gly, Gly-235
Ser, Leu-266
Met, and Gly-268
Ala) is sufficient to change the enzyme specificity from a
blood group A to a blood group B glycosyltransferase. To carry out a
systematic study, a synthetic gene strategy was employed to obtain
their genes and to allow facile mutagenesis. Soluble forms of a
recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically
characterized extensively for the first time. A functional hybrid A/B
mutant enzyme was able to catalyze both A and B reactions, with the
kcat being 5-fold higher for the A donor.
Surprisingly, even a single amino acid replacement in
glycosyltransferase A with the corresponding residue from
glycosyltransferase B (Arg-176
Gly) produced enzymes with
glycosyltransferase A activity only, but with very large (11-fold)
increases in the kcat and increased
specificity. The increases observed in kcat are
among the largest obtained for a single amino acid change and are
advantageous for the preparative scale synthesis of blood group
antigens.
Complex carbohydrates are becoming increasingly important for the key roles they play in cell signaling, molecular recognition, and many other biological processes (1). Specific glycosyltransferase enzymes are responsible for the synthesis of different disaccharide linkages in complex oligosaccharides by transferring a single monosaccharide unit from a nucleotide donor to the hydroxyl group of an acceptor saccharide. Cloned glycosyltransferase enzymes are powerful new tools in small to large scale synthesis of therapeutically significant oligosaccharides (2, 3). Glycosyltransferases are highly specific for the donor and acceptor substrates and have been grouped into families according to the type of sugar they transfer (4, 5). With a few exceptions, a different glycosyltransferase is required to synthesize each different glycosidic linkage, and it is estimated that there are over 100 different glycosyltransferases to account for all the documented oligosaccharides (6). Mammalian oligosaccharide chains are largely composed of nine monosaccharide units, which are transferred by glycosyltransferases to different acceptor structures, but there have been no extensive sequence similarities found among different glycosyltransferases. However, there are common structural features among mammalian transferases as they are all classified as type II integral membrane proteins, with a short amino-terminal cytoplasmic tail, a membrane-anchoring domain, a short proteolytically sensitive stem region, and a large catalytic domain that includes the carboxyl terminus (6).
The most homologous glycosyltransferase sequences are the A and B
glycosyltransferases of the human ABO blood group system (7). The
histo-blood group ABO(H) antigens are defined carbohydrate determinants
found on the surface of red blood cells and are largely responsible for
failure of mismatched blood transfusions. These ABO carbohydrate
antigens occur on other cell types and are important in cell
development, cell differentiation, and oncogenesis (8-10). Blood group
A individuals express
(1-3)N-acetylgalactosaminyltransferase (GTA),1 which catalyzes the transfer of
GalNAc from the donor UDP-GalNAc to the (O)H-precursor structure
Fuc
(1-2)Gal
-OR to give the A determinant
GalNAc
(1-3)[Fuc
(1-2)]Gal
-OR. Blood group B individuals express
(1-3)galactosyltransferase (GTB), which uses the same (O)H-structure but catalyzes the transfer of Gal from UDP-Gal to make
the B determinant Gal
(1-3)[Fuc
(1-2)]Gal
-OR (7, 11) (Fig.
1). Blood group O individuals do not express either
enzyme, and AB individuals express both (7).
The cDNA sequences of the GTA and GTB genes show that they are
highly homologous and that alteration of only four critical amino acid
residues (Arg-176 Gly, Gly-235
Ser, Leu-266
Met, and
Gly-268
Ala) is sufficient to change the enzyme specificity from a
blood group A to a blood group B enzyme (7, 12, 13). GTA and GTB
transfer to the same (O)H acceptor structure Fuc
(1-2)Gal
-OR, but
use different UDP-sugar donors. Therefore, it is assumed that these
four amino acids form the basis for the differential affinity of the A
and B glycosyltransferases for their nucleotide donors UDP-GalNAc and
UDP-Gal, respectively (13). In previous studies, transient gene
expression in human HeLa cells of combinatorial A-B transferase chimera
constructions which differed at the four critical amino acids suggested
that substitutions at positions 266 and 268 were crucial for the
nucleotide donor specificity of the enzyme (13). However, the authors
note that transient expression in HeLa cells may not be the true
representation of in vivo conditions and, moreover, does not
provide any mechanistic information about the enzymes. Therefore,
detailed kinetic analyses of purified enzymes are necessary to
determine the exact effects of the residue changes on substrate
recognition and catalytic activity (14).
There is a need for economical production of oligosaccharides, especially for the purpose of studying their biochemical function and assessing their potential in therapeutics or as diagnostic tools. Enzymatic oligosaccharide synthesis using glycosyltransferases proceeds regio- and stereoselectively and is less laborious and less costly compared with chemical synthesis. The limitation to the use of glycosyltransferases as synthetic tools is their scarcity from natural sources. The expression of recombinant glycosyltransferases in bacterial systems makes milligram scale synthesis by the enzymatic approach more practical (15).
Previously, we described the expression in Escherichia coli
of a soluble recombinant GTB and the kinetic characterization of its
acceptor specificity using synthetic analogs of the H disaccharide acceptor (16). In this paper, we report the chemical synthesis and
expression of DNA encoding GTA and a series of mutants. The sequence of
the synthetic DNA was designed to contain codons preferred for optimal
expression in E. coli and unique restriction sites to permit
systematic study of the specific effect of amino acid changes to
important residues. We also describe the extensive kinetic
characterization of wild-type GTA and a set of mutants where one, two,
and then three critical amino acid positions of GTA were substituted
with the corresponding amino acids found in GTB (Arg-176 Gly,
Gly-235
Ser, and Leu-266
Met). Wild-type GTA and the mutant
proteins were purified, kinetically characterized, and utilized in the
preparative scale synthesis of blood group A and B trisaccharides.
Oligodeoxyribonucleotides were synthesized using model 380A and 394 DNA/RNA synthesizers (Applied Biosystems). Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs and Life Technologies, Inc. All general molecular biology procedures were performed according to standard procedures (17). Gene design, extinction coefficients at 280 nm, and molecular weights of proteins were determined using the Wisconsin Sequence Analysis Software (Genetics Computer Group, Inc.). Pefabloc protease inhibitor (Boehringer Mannheim); UDP, UDP-GalNAc, and UDP-Gal (Sigma); UDP-[6-3H]GalNAc (10 Ci/mmol) and UDP-[6-3H]Gal (15 Ci/mmol) (American Radiolabeled Chemicals); Sep-Pak C18 reverse phase cartridges (Waters); Ecolite (+) liquid scintillation mixture (ICN); and Centriplus-10 protein concentrators (Amicon) were purchased commercially.
Construction of the Synthetic Glycosyltransferase Wild-type A Gene and MutantsThe ompA-GTA gene (1034 base pairs) was designed and synthesized as described previously for GTB (16). The mutant proteins were named based on their amino acid status at the four locations where GTA and GTB differ. The synthetic wild-type GTA (designated AAAA) and GTB (BBBB) genes were designed with unique restriction sites throughout the gene to facilitate mutagenesis. Glycosyltransferase mutants BAAA, BBAA, and BBBA were synthesized by digesting the GTB gene with KpnI/SphI and ligating in oligonucleotides to form the desired gene sequence. The DNA sequences of all four genes were confirmed on both strands.
Expression in E. coliPlasmids harboring the wild-type
ompA-GTA, and the mutant BAAA, BBAA, and BBBA genes were used to
transform E. coli TG-1 cells. To produce the recombinant
glycosyltransferase proteins, E. coli strains containing the
5 different plasmids were grown at 30 °C in M-9 minimal medium
supplemented with 0.4% casamino acids and 100 µg/ml ampicillin.
After 18-24 h the cultures were induced with 1 mM
isopropyl-1-thio--D-galactopyranoside and made up to 1 × TB (12 g of tryptone, 24 g of yeast extract, and 4 ml of
glycerol/liter of culture), a rich growth medium that maximizes cell
density. The cultures were harvested 48-64 h later, and periplasmic
extracts were prepared by a one-step osmotic shock. Cells from 2 liters of culture were resuspended thoroughly in 60 ml of ice-cold shock buffer (20 mM Tris-HCl, 1 mM EDTA (pH 7.2), 1.0 mM Pefabloc) and incubated on ice for 30-60 min. Proteins
were analyzed as described previously (16).
Soluble proteins
were purified from 60 ml of periplasmic extracts by precipitating with
ammonium sulfate. The 20-60% ammonium sulfate fraction was
resuspended in 1.5-3.0 ml of column buffer (20 mM Tris (pH
7.2), 1 mM dithiothreitol, 0.5 M NaCl) and
dialyzed extensively against the same buffer. The extracts were made up to 2 mM MnCl2, spun in a microcentrifuge for 5 min to remove any insoluble material and loaded onto a 10-ml
UDP-hexanolamine-Sepharose column at 0.4 ml/h with column buffer
containing 2 mM MnCl2 and washed extensively
until the A280 nm was at background levels. GTA
was eluted with column buffer containing 20 mM
MnCl2 and 2 mM UDP (18). Since 2 mM
UDP has a strong absorbance at 280 nm, the purified protein peak in the
column fractions was determined by taking 20 µl of each fraction and
measuring the protein concentration using the Bio-Rad Micro Protein
Assay. Pooled column fractions (3-4 ml) containing pure GTA enzyme
were concentrated to 600-1000-µl volumes using Centriplus-10 units,
dialyzed extensively against 20 mM sodium cacodylate (pH
6.8) containing 1 mM dithiothreitol before activity assays
or use in enzymatic synthesis. The protein concentration of all the
enzymes was determined using the Bradford method with bovine globulin as a standard (Bio-Rad).
For the kinetic analysis of GTA and GTB activity, Sep-Pak
radiochemical C18 assays were used with the hydrophobic
acceptor Fuc(1-2)Gal
-O-(CH2)7CH3
essentially as described previously (19-21). Reactions to measure
glycosyltransferase A activity were incubated at 37 °C for 20-60
min and carried out in a 33-µl total volume with 50 mM
sodium cacodylate buffer (pH 7.0), 20 mM MnCl2, 0.2 µCi of UDP-[6-3H]GalNAc, and 0.0005-0.2 µl of
purified GTA or mutant enzymes. Initial rate conditions were linear
under these conditions, where no more than 10-15% of substrate was
consumed. Six different concentrations of donor and acceptor were
employed generally covering the range from about 0.3 up to 8 Km for the substrate. Data were analyzed for a
general two-substrate system using Equation 1.
![]() |
(Eq. 1) |
The reactions to measure glycosyltransferase B activity were incubated at 37 °C for 30-120 min and carried out in 33 µl total volume with 50 mM sodium cacodylate buffer (pH 7.0), 20 mM MnCl2, 0.2 µCi of UDP-[6-3H]Gal, and 0.05-4.0 µl of purified GTA or mutant enzymes. Six different concentrations of donor and acceptor were employed and analysis carried out as described for the glycosyltransferase A activity.
One milliunit of activity is defined as the amount that catalyzes the conversion of 1 nmol of sugar transferred/min. The catalytic constant or turnover number, kcat, is the maximum number of substrate molecules converted to product per active site per unit time, obtained from vmax/[E].
The Preparative Scale Enzymatic Synthesis of the Blood Group A and B TrisaccharidesThe preparative scale enzymatic synthesis of
6.0 mg of blood group A trisaccharide was carried out in a reaction
mixture, which contained 6.0 mg (13.7 µmol) of precursor H
oligosaccharide Fuc(1-2)Gal
-O-(CH2)7CH3,
15 mg (22.4 µmol) of UDP-GalNAc, 100 mM sodium cacodylate
(pH 7.0), 5 mM MnCl2, 1 mg/ml bovine serum albumin, and 50 milliunits of recombinant GTA or BAAA enzyme in a total
volume of 0.8-1.0 ml. The reaction was incubated overnight at 37 °C
with GTA enzyme and UDP-GalNAc being added in two aliquots. The
progress of the reaction was monitored by thin layer chromatography with the product purified upon completion using two Sep-Pak
C18 reverse phase cartridges as described previously (15).
Similarly, BBBA was used in reactions with either UDP-GalNAc or
UDP-Gal, for the preparative scale synthesis of both A and B
trisaccharide products. The products were characterized by
1H NMR spectroscopy on a Varian Unity 500 spectrophotometer
at 500 Mhz.
The amino acid
sequence of membrane-bound human GTA deduced from cDNA (7, 12) was
used to redesign a 1034-base pair synthetic GTA gene to contain
E. coli preferred codons to maximize gene expression and
unique restriction sites to facilitate mutagenesis (Fig.
2). The putative transmembrane domain (amino acids
1-53) was replaced with the bacterial ompA secretory signal to target GTA into the periplasm as a soluble protein. A DNA segment encoding the
c-myc epitope and an affinity purification tail consisting of five histidine residues was added to the carboxyl terminus of the
GTA gene in front of two termination codons (Fig. 2). GTA (AAAA) and
GTB (BBBB) differ by only four essential amino acids (residues 176, 235, 266, and 268), and the mutant proteins were named based on their
amino acid status at the four locations. Only the unique restriction
enzymes sites in GTA are shown in Fig. 2, but other restriction sites
that differ between the GTA and GTB sequences are also present.
Expression and Purification
The soluble form of GTA and its
mutants were purified from the periplasm to apparent homogeneity and
characterized as described previously for GTB (16). The mutations in
BAAA (Arg-176 Gly) and BBAA (Arg-176
Gly and Gly-235
Ser)
enhanced total expression levels of the enzymes compared with that of
GTA and BBBA (Arg-176
Gly, Gly-235
Ser, and Leu-266
Met),
with a corresponding increased secretion of soluble recombinant protein
into the periplasm (data not shown). In terms of enzymatic activity,
the average yield of pure BAAA was 19 times higher than for GTA, due to
being more highly expressed in E. coli and having increased
catalytic activity.
Using a suitable range of six acceptor concentrations and six donor concentrations and then measuring the rate with all possible combinations of these substrate concentrations (i.e. 6 × 6 grid), the kinetic constants KA, KB, kcat, and kcat/Km were determined. In comparing wild-type glycosyltransferase A and B enzymes with each using their preferred donors, the kcat (maximum rate possible when both acceptor and donor is saturated) is similar. However, glycosyltransferase A has a Kia for its preferred donor UDP-GalNAc that is 5.5 times lower than the Kia of the B enzyme for its preferred donor UDP-Gal, which results in a higher specificity constant (kcat/Km) for the A enzyme (Table I).
|
Wild-type GTA and mutant BAAA and BBAA enzymes showed predominantly glycosyltransferase A activity (UDP-GalNAc) only. It was possible to kinetically characterize the small amounts of glycosyltransferase B activity (UDP-Gal) that was detectable in these glycosyltransferase A enzymes by using comparatively high concentrations of the recombinant enzymes in the assays for B activity. Kinetic analysis of the GTA enzyme using the B donor UDP-Gal shows that the wild-type A enzyme does not transfer the B donor (UDP-Gal) efficiently, largely because of a much lower kcat. Similarly, the decreased kcat also accounts for the lack of A activity (transferring of UDP-GalNAc) displayed by the B enzyme (Table I). KA and KB for GTA was similar for both UDP-GalNAc and UDP-Gal. Thus, the difference in the donor specificity of the glycosyltransferase A and B enzymes is largely due to a difference in kcat rather than Km values.
The single mutant BAAA (Arg-176 Gly) showed a very large 11-fold
increase in kcat and a 4-fold increase in the
specificity constant kcat/KA
compared to wild-type A enzyme (Table I). Although the single amino
acid change in BAAA was due to the substitution of residue Gly-176 from
the B enzyme for Arg in the A enzyme, this enzyme did not show any
additional ability to catalyze the B reaction, but instead showed an
increased ability to catalyze the A reaction.
A further additional amino acid change in the double mutant BBAA
(Arg-176 Gly, Gly-235
Ser) results in an enzyme with a 5-fold
increase in kcat compared with the wild-type A
enzyme. In contrast to BAAA, the specificity constant
kcat/KB is only increased slightly.
Despite two amino acid substitutions with the corresponding residues in
the B enzyme, BBAA is not able to catalyze either the A or B reaction
with the same specificity as either the wild-type A or B enzyme.
BBBA (Arg-176 Gly, Gly-235
Ser, and Leu-266
Met) has the
ability to catalyze both the A and B reactions and is a functional hybrid A/B enzyme. With UDP-GalNAc, BBBA has a 2-fold increase in
kcat, but also a larger KA
and KB, which results in lower specificity constants
compared with wild-type (Table I).
GTA
and BAAA were used in preparative scale synthesis of the blood group A
trisaccharide. Recombinant GTA (50 milliunits) was used in the
preparative scale synthesis of 6 mg of the blood group A trisaccharide
from the H disaccharide acceptor
Fuc(1-2)Gal
-O-(CH2)7CH3. The reaction was judged to be 50% complete after 6 h at 37 °C. The reaction was left for another 16-20 h, which resulted in 100% conversion to the A trisaccharide product. Aliquots of the reaction mixture were removed at different times, and the enzyme activity was
found to be relatively stable at 37 °C for 2 days. Under the preparative scale synthesis conditions, the mutant BAAA has the advantage of having a higher specific activity,
kcat, and
kcat/Km compared to wild-type
GTA. The use of the glycosyltransferase AB mutant BBBA with either
UDP-GalNAc or UDP-Gal donors in enzymatic synthesis also resulted in
100% conversion to the blood group A or B trisaccharide
determinants.
The structures of the synthetic A
(Fuc(1-2)[GalNAc
(1-3)]Gal
-O-(CH2)7CH3)
and B trisaccharide products
(Fuc
(1-2)[Gal
(1-3)]Gal
-O-(CH2)7CH3) were confirmed by 1H NMR. In the A trisaccharide product,
the terminal GalNAc is linked to Gal in an
1
3 linkage. The
resonance of the
-anomeric H of GalNAc was at
5.32 ppm
(J1,2 3.8 Hz) and that of the
-anomeric H of
Fuc was at
5.17 ppm (J1,2 3.8 Hz). For the B
trisaccharide product, the
-anomeric H of the terminal Gal linked to
the inner Gal in an
1
3 linkage results in the H-1 resonance at
5.30 ppm (J1,2 3.8 Hz) and the
-anomeric
H of Fuc at
5.24 ppm (J1,2 2.8 Hz) (22)
(data not shown).
In this paper, we describe the chemical synthesis and expression
of functional human GTA genes and its mutants using the synthetic gene
strategy. Purified recombinant GTA and its mutant BAAA from 1 liter of
E. coli had enzymatic activity equivalent to that
recoverable from 0.2 and 32 million liters of human blood group A sera
(19-21, 23-25), without the need for laborious multistep
chromatographic purification (23, 26). It is interesting to note that
the improved yield of active blood group A enzyme was accomplished without significantly altering the Km for the
acceptor, and, surprisingly, mutant BAAA showed 11-fold higher A
activity resulting from a single amino acid change of Arg-176 Gly.
The availability of large quantities of the recombinant enzymes has made possible the first systematic kinetic characterization of the
human blood group A and B enzymes. This allowed for an exact comparison
of steady-state kinetic parameters of wild-type A enzyme, the mutants,
and the B enzyme for both UDP-GalNAc and UDP-Gal donors.
Although GTA and GTB differ by only four amino acids and use the same acceptor, the alteration of these amino acids affects the binding of both the UDP-sugar donor and the acceptor. Kinetic characterization revealed that alteration of even one of the four amino acids that differ between these two enzymes may affect the Km of both the acceptor and the donor substrates. The Km for the acceptor also differed depending on whether UDP-GalNAc or UDP-Gal was used as the donor (Table I). GTB has a KB for the donor UDP-Gal higher than the KB that GTA has for the donor UDP-GalNAc, which suggests that the binding of GTB to UDP-Gal is weaker than the binding of GTA to UDP-GalNAc, with the corresponding kcat values being similar.
The major difference in the utilization of the alternate donors by the
glycosyltransferase A and B enzymes is largely due to a difference in
kcat rather than Km. For GTA
the kcat for UDP-Gal is only 0.4% that of
UDP-GalNAc. GTA had an apparent Km for UDP-Gal
similar to the Km for UDP-GalNAc, indicating that
the enzyme was able to bind both donors, but the significantly
decreased kcat for UDP-Gal showed that it was
not able to readily catalyze the transfer. Similarly, the lack of GTB
activity observed for BAAA (Arg-176 Gly) and BBAA (Arg-176
Gly,
Gly-235
Ser) was not necessarily due to an increased Km, but also largely due to a decreased
kcat.
The mutant BAAA was similar to GTA in that it possessed essentially
only GTA activity. Alteration of GTA by the single mutation (Arg-176
Gly) did not alter the donor substrate specificity but did alter
the kinetic properties of the enzyme by increasing kcat 11-fold, as well as increasing the
specificity constant, with respect to both the acceptor
(kcat/KA) and the donor UDP-GalNAc (kcat/KB) (Table
I). It is surprising that substitution of the corresponding residues
from GTB into GTA in these two mutant enzymes did not result in any
additional glycosyltransferase B activity but instead produced enzymes
with glycosyltransferase A activity higher than that found in wild-type
GTA. Indeed these are among the largest increases in
kcat observed for a single amino acid
change.
It is interesting to note that transient expression studies in HeLa cells suggest BAAA is half as active as wild-type A enzyme (13). In contrast, detailed kinetic analysis of the purified enzymes illustrates that it is essential to determine the exact effects of residue changes on substrate recognition and catalytic activity. The transient expression studies provide no mechanistic information (14).
The triple mutant BBBA (Arg-176 Gly, Gly-235
Ser, and Leu-266
Met) was a functional hybrid A/B enzyme, which was used to
synthesize both the blood group A and B trisaccharides, since it had
significant A and B activity. The structural difference between
Gal
and
GalNAc is spatially significant as the 2-deoxy-2-acetamido group
in
GalNAc is larger than the 2-hydroxyl group in
Gal. Glycosyltransferase AB enzymes exist in nature and may provide an
explanation for the rare cis-AB phenotype observed (27).
The maximum rate possible when both substrates are saturated
(kcat) is often affected by rate-limiting
substrate binding or product release steps. The specificity constant
(kcat/Km), which combines the
effects of both rate and binding, was greatly increased in BAAA for
both the acceptor and the donor. Generally, irrespective of the
mechanism, the major factor governing specificity is the stability of
the enzyme-bound transition state, which exists during the conversion
of the enzyme-bound substrate to product. It is possible that the
removal of the Arg side chain at position 176 in mutant BAAA stabilizes
the transition state with the donor GalNAc more effectively,
resulting in the higher specificity observed.
Our results suggest that alteration of the four critical amino acids that differ between GTA and GTB affected the kinetic constants of both the acceptor and the donor, possibly due to topologically close binding of the donor and acceptor. Mutations at residue 176 in BAAA appear to have little effect on the binding of the acceptor, but do affect the enzyme turnover, as this mutant showed a very large increase in kcat. Mutations at residues 176 and 235 in BBAA showed a 5-fold increase in kcat and weaker acceptor binding, and the binding of the donor was not greatly affected. These results suggest a role in enzyme turnover for residue 176. Residue 235 could affect the binding of the acceptor, and segments around residues 266 and 268 could be most critical for binding of the nucleotide sugar donor.
Recombinant GTA and BAAA enzymes were used in the preparative scale synthesis of the blood group A trisaccharide, with BAAA having the advantage of having a 11-fold higher kcat compared with wild-type GTA. A further systematic mutagenesis of GTA and GTB may lead to glycosyltransferase enzymes with even more significant increases in catalytic activity.
Very little is known about structure/function relationships in glycosyltransferases. With a few exceptions, there is very little nucleotide or amino acid sequence similarity among glycosyltransferases even if they use the same acceptor or nucleotide donors. There are no x-ray crystal structures of mammalian glycosyltransferases known, and the large scale fermentation of E. coli to produce unglycosylated glycosyltransferases may produce enough protein for crystallization and subsequent three-dimensional structure determination. The availability of recombinant GTA, GTB, and their mutants can form the framework for further genetic engineering of new glycosyltransferases, allowing investigation into structure and function relationships to produce new glycosyltransferase enzymes as synthetic tools and diagnostic reagents.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y11891[GenBank].
We thank Dr. Ole Hindsgaul for providing the acceptor substrates, helpful discussions throughout this work, and reading of the manuscript. We also thank Dr. A. Otter for the NMR spectra, D. Bilous for the synthesis of the oligonucleotides, J. Michniewicz for assistance with DNA sequence analysis, D. Pope for general assistance, and Dr. N. M. Young for reading of the manuscript.