Biochemical studies of inositol: N -acetylglucosaminyltransferase involved in mycothiol biosynthesis in Corynebacterium diphtheria

Article abstract of DOI:10.1039/c7ob00555e

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.

Full text of DOI:10.1039/c7ob00555e

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DOI: 10.1039/C7OB00555E
Biochemical Studies of Inositol N-Acetylglucosaminyltransferase Involved in
Mycothiol Biosynthesis in Corynebacterium diphtheria
Yuchuan Guo,a,b Lizhen Wang,a Jiatong Guo,b Guofeng Gu,a,* and Zhongwu Guob,*
a National Glycoengineering Research Center and Shandong Provincial Key Laboratory of Carbohydrate Chemistry
and Glycobiology, Shandong University, 27 Shanda Nan Lu, Jinan 250100, China
b Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida 32611, United States
* Corresponding author E-mails: guofenggu@sdu.edu.cn; zguo@chem.ufl.edu
Abstract
Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and
it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P)
α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase
involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as
CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc
to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product
3-phospho-1-D-myo-inositol-2-acetamido-2-deoxy-α-D-glucopyranoside. CdMshA was shown to
accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors,
such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors,
such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate
selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as
substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino
acid residues N28, K81 and R157 were essential for its enzymatic activity.
Key words: Mycothiol, Inositol N-acetylglucosaminyltransferase, Corynebacterium diphtheria,
substrate selectivity, site-directed mutagenesis

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Introduction
Mycothiol (MSH), 1-D-myo-inositol 2-(N-acetyl-L-cysteinyl)amino-2-deoxy-α-D-glucopyranoside,
is the principal low molecular thiol in actinobacteria and is the functional equivalent of glutathione
found in Gram-negative bacteria and most eukaryotes.1, 2 Similar to glutathione, mycothiol plays a
pivotal role in protecting bacteria against oxidative stresses and in the detoxication of xenobiotics.3
In mycobacteria where MSH has been extensively investigated, MSH-deficient mutants exhibited
increased sensitivities to oxidative stresses, alkylating agents, and a broad range of antibiotics,4, 5
suggesting the significance of MSH in mycobacterial survival and pathogenicity.6 Consequently,
targeted disruption of MSH biosynthesis in Mycobacterium tuberculosis Erdman produced no
viable bacterial clones, indicating that MSH is essential for M. tuberculosis Erdman.6 In addition,
it has been demonstrated that MSH was important for Corynebacterium glutamicum resistance to
alkylating agents, glyphosate, ethanol, antibiotics, heavy metals, and aromatic compounds7 and
that increasing the intracellular MSH content in C. glutamicum could improve its robustness to
various stresses.8 Therefore, the MSH biosynthetic pathway represents a promising target for the
development of new antibacterial therapeutics or novel agents that can improve the efficacies of
current antibiotics.9, 10
As shown in Scheme 1, MSH biosynthesis consists of five enzymatic steps3, 11 mediated respectively
by an inositol N-acetylglucosaminyltransferase (GlcNAc-T) (MshA),12-14 a phosphatase (MshA2),
a GlcNAc deacetylase (MshB),15-20 an ATP-dependent ligase (MshC),21-24 and an acetyltransferase
(MshD).25, 26 Except for MshA2, the other four enzymes from different bacterial species have been
identified. As outlined, the first dedicated step for MSH biosynthesis is MshA-mediated transfer of
a GlcNAc unit from α-GlcNAc uridine diphosphate (UDP-GlcNAc) to 1L-myo-insitol-1-phosphate
(1L-Ins-1-P), which affords 3-phospho-1-D-myo-inositol-2-acetamido-2-deoxy-α-D-glucopyranoside
(GlcNAc-Ins-3-P). An MshA from C. glutamicum (CgMshA) was studied by X-ray crystallography
which helped the understanding of this enzyme.12 However, in general, information about MshA,
and other enzymes involved in MSH biosynthesis as well, is limited and more detailed studies on
these enzymes are necessary to gain insights into their functions and application.

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Scheme 1. The MSH biosynthetic pathway
We described herewith the expression, purification, and characterization of a novel MshA from C.
diphtheria, named as CdMshA. The biological properties of this enzyme, including its reaction
kinetics and substrate specificity, were studied in detail employing commercial sugar donors and
chemically synthesized inositol and sugar derivatives as glycosyl acceptors. Moreover, site-directed
mutagenesis of CdMshA was performed to investigate its structure-enzymatic activity relationship.
Results and Discussion
Expression and purification of CdMshA. The MshA-encoding gene mshA was derived from C.
diphtheria, synthesized by Sangon biotech, and ligated into the NdeI/XhoI sites of the pET-22b
expression vector. Thereafter, the recombinant plasmid carrying mshA was transferred into E. coli
o
BL21 (DE3), and the transformant was cultured in LB medium containing ampicillin at 37 C
overnight and then transferred into a fresh medium in a 1:100 (v/v) ratio. Enzyme expression was
induced upon the addition of IPTG after the cell culture reached an optical density (OD) value of
0.60~0.80 at 600 nm. Following continuous cultivation at 16 oC for 12 h, cells were harvested and
disrupted by ultrasonication. The cell lysate was centrifuged at 4 oC and 12000 rpm for 30 min to
obtain the crude enzyme in the supernatant, which was further purified to homogeneity by
Nickel-chelation chromatography. The purified enzyme showed a single band corresponding to
~50 kDa in SDS-PAGE (Figure S1), which was in accordance with the theoretical mass prediction
(47,416 Da) of CdMshA.

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Enzymatic activity, substrate specificity, and reaction kinetics of CdMshA. The natural substrate
of MshA, 1L-Ins-1-P, was synthesized by a reported procedure (Scheme 2)27, 28 and used to assess
the enzymatic activity of purified CdMshA to catalyze the transfer of GlcNAc to inositol. First,
myo-inositol was converted into racemic (±)-1,28-30 which was then resolved in two steps, including
acylation using (-)-menthyl chloroformate and separation of the resultant diastereomers through
recrystallization from methanol to obtain isomer (+)-2, followed by its saponification to produce
enantiomerically pure (+)-1. Thereafter, (+)-1 was phosphorylated smoothly to give (+)-3 that was
finally deprotected to provide 1L-Ins-1-P. The analytical data of all synthetic intermediates and the
final product agreed well with their structures and the reported data.28-31
Scheme 2. Synthesis of 1L-Ins-1-P
Assays of the enzymatic activity of CdMshA were performed in Tris-HCl buffer (50 mM, pH 8.0)
containing 1L-Ins-1-P (0.5 mM) and UDP-GlcNAc (1 mM) in a 1:2 ratio, and the concentration of
CdMshA was 60 μg/mL. The reaction mixture was kept at 37 oC, and its aliquots were taken at 1,
o
o
2 and 4 h, respectively, and quenched with preheated (60 C) acetonitrile and incubation at 60 C
for 10 min. After the reaction mixtures were cooled with ice and centrifuged (13,000 × g, 10 min)
to remove the protein, they were subjected to reversed-phase HPLC analysis. As the reaction product,
GlcNAc-Ins-3-P, did not have UV absorptions, we analyzed the reaction mixtures by observing the
consumption of UDP-GlcNAc and the generation of UDP. As depicted in Figure 1, after 1, 2 and 4
h of incubation, 15%, 32% and 48% of UDP-GlcNAc was transformed into UDP in the presence
of CdMshA, while no conversion or decomposition of UDP-GlcNAc was observed under the same

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conditions without CdMshA. These data suggested that the conversion rates of 1L-Ins-1-P under the
specified conditions were 30%, 64%, and 96%, respectively. Finally, the product was isolated from
the reaction mixture by ion-exchange HPLC to verify the formation and yields of GlcNAc-Ins-3-P,
and its structure was characterized with MS and NMR spectroscopy.12 For example, the coupling
constant of the anomeric proton (JH1-2 = 3.6 Hz) of its GlcNAc confirmed the α-configuration.
These results proved unambiguously the enzymatic activity of CdMshA to catalyze GlcNAc
transfer to 1L-Ins-1-P. Eventually, CdMshA was used to realize milligram-scale synthesis of
GlcNAc-Ins-3-P, which offered a 95% isolated yield.
Figure 1. HPLC results of CdMshA-catalyzed reaction between UDP-GlcNAc and 1L-Ins-1-P for 1, 2, and
4 h, respectively. The reaction was carried out in Tris-HCl buffer (50 mM, pH 8.0) containing UDP-GlcNAc
(1 mM) and 1L-Ins-1-P (0.5 mM), and the concentration of CdMshA was 60 μg/mL. HPLC conditions: C18
column (4.6 x 150 mm), 1 mL/min flow rate, gradient eluents using buffer A (2 mM tetrabutylammonium
hydroxide in water at pH 6.5) and buffer B (10 mM tetrabutylammonium hydroxide and 77 mM KH2PO4 in
50% aqueous methanol at pH 5.5) and UV detection at 260 nm.
After the enzymatic activity of CdMshA was verified, we continued to investigate its substrate
specificity. In this case, all reactions were carried out and analyzed under the same conditions
described above. First, we used the proved glycosyl acceptor 1L-Ins-1-P to probe various glycosyl
donors, including UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, as enzymatic substrates and
found that they were not accepted by CdMshA. Then, using UDP-GlcNAc as the glycosyl donor,
we examined the reactivity of myo-inositol, 1D-Ins-1-P derived from (±)-1 by the same method
outlined in Scheme 2 with (+)-menthyl carbonate as the resolution reagent, and a series of sugar

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phosphates including glucose-6-phosphate, galactose-6-phosphate, mannose-6-phosphate,
glucose-1-phosphate in the presence of CdMshA. We found that these compounds were not
accepted by CdMshA as glycosyl acceptors either. The results demonstrated the rigid substrate
selectivity of CdMshA, which was in agreement with previous reports.12, 13
The biochemical properties of CdMshA were further characterized through studying its reaction
kinetics and the influence of various conditions, such as temperature, pH and metal cations, on its
enzymatic activity. First, we examined the reaction of UDP-GlcNAc and 1L-Ins-1-P using CdMshA
in Tris-HCl buffer (50 mM, pH 8.0) at various temperatures. As depicted in Figures 2A, CdMshA
o
showed the maximal activity at 40 C, but it could tolerate a wide range of temperature, as about
90% of the catalytic activity was retained from 20 to 50 oC. Next, we studied the influence of pH
on this enzymatic reaction. As shown in Figures 2B, CdMshA exhibited good activity (>80%) at
the pH ranging from pH 7 to 10, and the maximal activity was observed at pH 8.0.
Figure 2. The influences of temperature (A), pH (B), and metal cations (C) on the activity of CdMshA
It was also discovered that divalent metal ions were not strictly necessary for the catalytic activity
of CdMshA (Figure 2C), as the enzyme exhibited good activity in the absence of a divalent metal
cation. In this respect, CdMshA was similar to other reported GT-4 family glycosyltransferases,
such as L-malate α-N-acetylglucosaminyltransferase BshA involved in bacillithiol biosynthesis.32
However, metal cations did exhibit some influences on the enzymatic activity of CdMshA. For
example, Mg2+, Ca2+, and K+ could slightly enhance the activity of CdMshA, whereas Ag+, Ni2+,
Fe2+, Cu2+, Hg2+, Zn2+, and Mn2+ demonstrated minor to significant inhibitions on CdMshA. These

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findings were consistent with a previous report32 of slight activity enhancement of BshA by Mg2+
and an observation made with the crystal structure of C. glutamicum MshA, namely that MshA
contains Mg2+ but the metal cation is not located at the active site.12
After the optimal reaction conditions for CdMshA (Tris-HCl buffer, pH 8.0, 40 oC) were established,
they were employed to examine the reaction kinetics of this enzyme and determine the Km and
Vmax values for both substrates. Initial reaction velocities for 1L-Ins-1-P and UDP-GlcNAc were
obtained with varied 1L-Ins-1-P concentrations/saturated UDP-GlcNAc and varied UDP-GlcNAc
concentrations/saturated 1L-Ins-1-P, respectively. The data were utilized to calculate the Michaelis
constants (Km) for 1L-Ins-1-P and UDP-GlcNAc, which were 0.485 ± 0.049 mM and 0.185 ±
0.047 mM (Figure S2), respectively. The Vmax values for 1L-Ins-1-P and UDP-GlcNAc were 0.437
± 0.022 and 0.328 ± 0.028 nmol Min-1, and the Kcat values were 6.907 ± 0.347 Min-1 and 5.184 ±
0.442 Min-1, respectively. The Km value of CdMshA for UDP-GlcNAc was comparable to those of
the crude MshAs derived from M. smegmatis (0.17 ± 0.02 mM) and C. glutamicum (0.208 ± 0.017
mM).12, 13 However, the Km value of CdMshA for 1L-Ins-1-P was about two times higher than those
of the crude MshAs from M. smegmatis (0.15 ± 0.01 mM) and C. glutamicum (0.236 ± 0.013 mM).
These results indicated that CdMshA had a slightly lower affinity towards 1L-Ins-1-P than other
similar enzymes.
Mutagenic studies of CdMshA. To identify the key amino acids responsible for the enzymatic
activity of CdMshA, we probed site-directed mutagenesis of this enzyme. The target amino acids
were selected according to a reported crystal structure of CgMshA,12 whose amino acid sequence
was ca. 72% identical to that of CdMshA. It was reported that MshA follows an ordered sequential
kinetic mechanism with UDP-GlcNAc binding first. After binding with UDP-GlcNAc, MshA has
a large conformational change to bring UDP-GlcNAc and 1L-Ins-1-P into close proximity.12 The
crystal structure of CgMshA also revealed that at its active site (Figure 3A), the side chains of K78,
R154, T134, and Y110 could form hydrogen bonds with the phosphate group of 1L-Ins-1-P and
that the M24, N25 and D20 residues had polar interaction with the 3-OH, 4-OH, and 5-OH groups
of 1L-Ins-1-P, respectively. The 2-OH group in 1L-Ins-1-P pointed into a small pocket generated

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by the side chains of M24, Y110, T134, and UDP-GlcNAc.12 Our analysis of MshAs from various
species further suggested that amino acids D20, M24, N25, K78, Y110, T134, and R154 were
highly conserved (Figure S3). Based on the crystal structure of MshA and the above analyses, we
constructed a homology model for the active site of CdMshA (Figure 3B). This model indicated
that its D23, M27, N28, K81, Y113, T137, and R157 residues were correspondent to the conserved
amino acids of MshAs identified above. Therefore, in this study, we mutated these amino acids to
alanine and examined the activities of the resultant mutant enzymes.
Figure 3. Crystal structure of the active center of CgMshA (A),12 and the homology model of CdMshA (B).
Homology modeling of CdMshA was carried out using the Phyre2 program, and the images were created
by the software Pymol-1.4.1.
Site-directed mutation of the CdMshA gene was achieved by standard protocols,33 and the mutant
enzymes were expressed and purified as described above. Assays of the enzymatic activities of the
CdMshA mutants were carried out under the above-mentioned optimal reaction conditions, and the
results were depicted in Figure 4. Compared to wild type CdMshA, the D23A, M27A, and Y113A
mutants exhibited a mild reduction in catalytic activity, while the T137A mutant retained only ca.
30% of activity. In contrast, N28A, K81A, and R157A mutations abolished the enzymatic activity
almost completely (<5% of activity retained). Clearly, N28, K81, and R157, as well as T137, played
a critical role in the enzymatic activity of CdMshA. According to the models depicted in Figure 3,
K81/R157 and N28 should form hydrogen bonds with the phosphate and the 4-OH groups of

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1L-Ins-1-P, respectively, which would help keep 1L-Ins-1-P in proper orientation at the active site
of CdMshA and hold its 3-OH group close to the pyrophosphate group of UDP-GlcNAc. This
should be critical for the enzymatic reaction to occur. T137 could also form a hydrogen bond with
the phosphate group of 1L-Ins-1-P and, in the meantime, work together with M27 and Y113 to
create a small pocket to accommodate the axial 2-OH group of 1L-Ins-1-P and limit its freedom to
facilitate the reaction. Comparably, the roles of D23 and M27 were relatively insignificant, as the
activity of CdMshA was only mildly affected after mutation at these sites. The requirement of
multiple hydrogen bonds formed between CdMshA and the phosphate and hydroxyl groups of
1L-Ins-1-P for the enzyme to function properly and the tight space at the active center of CdMshA,
as proposed in the model, may also explain the rigid substrate selectivity of CdMshA.
Figure 4. Relative enzymatic activities of various CdMshA mutants
Finally, employing UDP-GlcNAc as the glycosyl donor, we assessed the activities of these mutant
enzymes, especially D23A, M27A, Y113A and T137A, to incorporate unnatural glycosyl acceptors
e.g., 1D-Ins-1-P, myo-inositol, glucose-6-phosphate, galactose-6-phosphate, mannose-6-phosphate
and glucose-1-phosphate. HPLC analysis of the reaction mixtures revealed no UDP production,
indicating that the mutant enzymes did not use these compounds as substrates. Thus, mutagenesis
at the identified sites could not relax the substrate specificity of CdMshA.
Conclusion
In this research, we have accomplished the expression, purification, and characterization of a new
MshA from C. diphtheria and named it as CdMshA. We have demonstrated that CdMshA could

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effectively catalyze the initial step of mycothiol biosynthesis, that is, transferring GlcNAc from
UDP-GlcNAc to 1L-Ins-1-P to generate GlcNAc-Ins-3-P, which was isolated and characterized.
CdMshA exhibited relatively rigid substrate selectivity, as it did not accept a number of common
glycosyl donors and acceptors including 1D-Ins-1-P, a diastereomer of 1L-Ins-1-P. Our studies
further revealed that CdMshA tolerated a relatively wide range of pH (7-10) and temperature (20
o
to 50 C). Similar to MshAs from other bacteria, the enzymatic activity of CdMshA did not need
divalent metal cations but certain cations like Mg2+, Ca2+ and K+ could slightly enhance its activity.
However, kinetics analysis indicated that compared to other MshAs, CdMshA exhibited a slightly
lower affinity towards 1L-Ins-1-P. Furthermore, mutagenic studies on CdMshA combined with
molecular modeling of its active center revealed that N28, K81, T137 and R157 in its peptide
sequence are the key amino acids for the enzymatic activity. This study has provided some detailed
insights into CdMshA, which should be very helpful for the design and development of inhibitors
against the enzyme and other potential applications.
Materials and Method
Materials and general methods. Restriction endonucleases were purchased from New England
Biolabs. Resins and columns used for Nickel chelation chromatography were obtained from GE
Healthcare. PCRs were performed with Q5® High-Fidelity DNA Polymerase that was obtained
from NEB. The gel extraction, PCR purification, and plasmid extraction kits were purchased from
Sangon Biotech. Other reagents and chemicals were purchased from commercial sources and used
without further purification or treatment if not specified otherwise.
Bacterial strains and growth condition. Bacterial strains and plasmids used in this study are listed
in Table S1. E. coli was cultured in shaking LB broth (180 rpm), and if needed, ampicillin (100
μg/mL) was used as the antibiotic.
DNA isolation and manipulation. General DNA manipulations, transformations, and agarose gel
electrophoresis were carried out by standard protocols. Restricted enzyme digestion, ligation, and
plasmid purification were performed in accordance with the manufacturers’ instructions. DNA

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sequencing, gene synthesis, and primer synthesis were carried out by Sangon Biotech, and primers
used in this study are listed in Table S1.
Expression and purification of recombinant proteins. After recombinant pET-22b-cdmsha genes
were transformed into the E. coli BL21 (DE3) host, the recombinant bacterial strains were cultured
in LB broth at 37 oC to an OD600 of 0.6~0.8. The culture was cooled to 16 oC, and then isopropyl
β-D-1-thiogalactopyranoside (IPTG, 0.4 mM) was added to induce protein expression, while the
cells were allowed to grow for additional 12 h. The cells were harvested through centrifugation,
and then re-suspended in the binding buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8.0, four times
the original volume), followed by disruption with sonication and centrifugation at 12,000 rpm and
4 oC for 30 min to remove cell debris. Protein purification was achieved via affinity chromatography
using a Nickel chelation column. Accordingly, after cell supernatants obtained above were loaded
onto His trap columns, the columns were washed first with Tris-HCl buffer and then with 100 mM
imidazole in buffer (50 mM Tris-HCl containing 500 mM NaCl) to elute target proteins carrying a
His-tag. Protein samples were analyzed with 10% SDS-PAGE and visualized by Coomassie brilliant
blue staining. Pure proteins were dialyzed three times against Tris-HCl buffer (50 mM, pH 8.0) at
o
4 C and finally concentrated by ultrafiltration (Millipore). Protein concentration was determined
using the Bradford protein assay kit following the manufacturer’s instructions with bovine serum
albumin (BSA) as standard. The proteins were stored at -80 oC.
Evaluation of the enzymatic activity of CdMshA. To the solution of UDP-GlcNAc (1 mM) and
1L-Ins-1-P (0.5 mM) in Tris-HCl buffer (50 mM, pH 8.0, 300 µL) was added CdMshA (60 μg/mL).
After the mixture was incubated at 40 oC, 50 µL aliquots were taken from the mixtures at 1, 2, and
4 h and quenched by adding the same volume of heated acetonitrile (60 oC) and then incubation at
60 oC for 10 min. After the resultant samples were cooled with ice and centrifuged (13,000 × g，10
min) to remove insoluble proteins, they were subjected to HPLC analysis. HPLC conditions were:
C18 column (4.6 x 150 mm); 10 μL sample for each injection; UV monitor at 260 nm;13 1 mL/min
flow rate; gradient eluent [0 to 1 min: 100% buffer A; 1 to 11 min: 75% buffer A and 25% buffer B;
11 to 31 min: 100% buffer B; thereafter: 100 % buffer A. Buffer A: tetrabutylammonium hydroxide

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(Bu4NOH) aqueous solution (2 mM, pH 6.5); buffer B: Bu4NOH solution (10 mM, pH 5.5) in 50%
aqueous methanol containing 77 mM of KH2PO4]. Under these conditions, the retention times for
UDP-GlcNAc and UDP were 20.2 and 21.5 min, respectively.
Enzymatic synthesis of GlcNAc-Ins-3-P.12 To a solution of UDP-GlcNAc (10 mM) and 1L-Ins-1-P
(10 mM) in Tris-HCl buffer (50 mM, pH 8.0, 0.5 mL) was added CdMshA (100 μg). The reaction
progress was monitored by HPLC according to the above-described protocol. When the reaction
was complete (in ca. 4 h), the reaction product, GlcNAc-Ins-3-P, was isolated by HPLC using a 1
mL-Mono Q ion-exchange column following a protocol similar to that previously reported.12 Thus,
the reaction mixture was injected into the Mono Q column and fractionated using a programmed
gradient eluent: 0-5 min with 100% buffer A, 20 min with 65% buffer A and 35% buffer B, 25-30
min with 100% buffer B, 33 min with 100% buffer A, where buffer A is 20 mM of aq. ammonium
bicarbonate solution and buffer B is 600 mM of aq. ammonium bicarbonate solution. The flow rate
was 1 mL/min, monitored at 260 nm. The resultant fractions (1 mL each) were then treated with
alkaline phosphatase and assayed for inorganic phosphate using malachite green phosphate assay.
Fractions that were found to contain phosphate but did not correlate to the peak at 260 nm were
1
pooled and lyophilized to afford GlcNAc-Ins-3-P (2.2 mg, 95%) as a white solid. H NMR (600
MHz, D2O, with DHO at δ 4.78 ppm as internal reference): δ 5.12 (d, 1 H, J = 3.6 Hz, H-1GlcNAc),
4.38 (br s, 1 H, H-2Ins), 3.96-3.91 (m, 2 H, H-2GlcNAc, H-3Ins), 3.91-3.85 (m, 2 H, H-5,6aGlcNAc),
3.82 (t, 1 H, J = 9.6 Hz, H-3GlcNAc), 3.81-3.74 (m, 3 H, H-6bGlcNAc, H-5,6Ins), 3.61 (br d, 1 H, J =
13
10.2 Hz, H-1Ins), 3.50 (t, 1 H, J = 9.6 Hz, H-4GlcNAc), 3.35 (t, 1 H, J = 9.6 Hz, H-4Ins); C NMR
(150 MHz, D2O): δ 174.26, 99.46, 79.20, 74.66, 74.01, 72.38, 71.78, 71.66, 71.03, 70.80, 69.83,
62.37, 53.78, 21.85; 31P NMR (242 MHz, D2O): δ 2.50. HR ESI-MS m/z: calcd for C14H25NO14P-1
(M - H)-1, 462.1018; found, 462.0998.
Evaluation of the substrate selectivity of CdMshA. These assays were performed in triplicate
under the same conditions used for the enzymatic activity assays of CdMshA. With 1L-Ins-1-P as
the glycosyl receptor, the glycosyl donors evaluated included UDP-GlcNAc, UDP-Gal, UDP-Glc,
UDP-GalNAc, and UDP-GlcA. With UDP-GlcNAc as the glycosyl donor, the glycosyl receptors

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tested included 1L-Ins-1-P, 1D-Ins-1-P, myo-inositol, glucose-6-phosphate, galactose-6-phosphate,
mannose-6-phosphate, and glucose-1-phosphate.
Evaluation of the influences of pH, temperature and metal cation on the activity of CdMshA. To
evaluate the influence of pH on the activity of CdMshA, the enzymatic reaction was examined in a
buffer containing 6.01 g/L of citric acid, 3.89 g/L of KH2PO4, 1.77 g/L of boric acid, and 5.27 of
g/L barbitone with the pH values adjusted in a range of 4 to 12 using NaOH. The concentrations of
UDP-GlcNAc, 1L-Ins-1-P, and CdMshA utilized were the same as that in above experiments. The
o
reaction was kept at 37 C for 30 min before it was quenched and subjected to HPLC analysis by
the protocols described above. To study the influence of temperature on the activity of CdMshA,
the reaction conditions described for the activity assays were used, but the reaction was carried out
o
at varied temperatures including 20, 25, 30, 35, 40, 45, 50 and 55 C. After the reaction mixture
was kept at certain temperature for 30 min, it was quenched and analyzed by HPLC as described
above. Similar experimental setup and conditions were also employed to evaluate the influence of
mental ions on the activity of CdMshA, and cations tested included Na+, K+, Ag+, Ni2+, Fe2+, Cu2+,
Hg2+, Zn2+, Mn2+, Mg2+, and Ca2+ and their concentration was set at 1 mM. The reaction was kept
at 37 oC for 30 min before it was quenched and analyzed.
Analysis of the kinetics of CdMshA-catalyzed reactions. These reactions were carried out under
o
the optimized conditions described for the activity assays (Tris-HCl buffer, pH 8.0, 40 C, and 60
μg/mL of CdMshA) employing varied concentrations of UDP-GlcNAc or 1L-Ins-1-P. In one set of
experiments, the concentration of 1L-Ins-1-P was 1 mM, while the concentration of UDP-GlcNAc
varied from 0.05 to 1 mM. In the other set of experiments, the concentration of UDP-GlcNAc was
kept at 1 mM, and the concentration of 1L-Ins-1-P varied from 0.05 to 1 mM. The reactions were
quenched at 30 min and analyzed by HPLC as described. The data obtained were used to calculate
initial velocities and determine the Michaelis constants (Km) and maximal velocities (Vmax) with
GrapPad Prism. All assays were performed in triplicate.
Mutagenesis of MshA and evaluation of the enzymatic activity of the mutants. Site-directed
mutagenesis of MshA with its D23, M27, N28, K81, Y113, T137 and R157 amino acids substituted

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with an alanine residue, respectively, was performed with an Easy Mutagenesis kit according to
standard protocols.33 Briefly, forward and reverse primers were designed, synthesized, and utilized
to replace the designated residue (Table S1) with the pET-22b-mshA vector as the template. PCR
reactions were performed to obtain the amplified fragments, which were then treated with Dpn I
enzyme to remove the template and transformed into E. coli DH5α. The mutant plasmids were
extracted from E. coli and sequenced to confirm the mutations in mshA gene. The mutant proteins
were expressed and purified (SDS-PAGE results in Figure S4) by the above-mentioned protocols.
The activities of these mutant enzymes were assessed under the same conditions described for the
o
wild type enzyme. The reactions were kept at 40 C for 30 min before they were quenched and
analyzed by HPLC. Similarly, the selectivity of these mutant enzymes for glycosyl acceptors were
also assessed using UDP-GlcNAc as glycosyl donor. The glycosyl acceptors examined included
1D-Ins-1-P, myo-inositol, glucose-6-phosphate, galactose-6-phosphate, mannose-6-phosphate, and
glucose-1-phosphate as glycosyl acceptor. All these assays were performed in triplicate.
Synthesis of (+)-2. To a solution of (±)-1 (200 mg, 0.857 mmol) in the mixture of DCM (2 mL)
and pyridine (2 mL) were added triethylamine (TEA, 0.5 ml) and (-)-methyl chloroformate (0.5
ml). The solution was stirred at rt overnight. After TLC showed the completion of reaction, the
solvent was removed, and the residue was purified by column chromatography (petroleum ether
and ethyl acetate 15:1) to afford the product as colorless syrup. This product was then dissolved in
MeOH and allowed to stand at 2 oC overnight. The resultant crystal was filtered and washed with
cold methanol to give (+)-2 (80 mg, 21%). 1H NMR (600 MHz, CDCl3): δ 7.36-7.25 (m, 5 H, Ph),
5.14 (dd, 1 H, J = 4.2, 2.4 Hz, H-2), 4.73 (s, 2 H, CH2Ph), 4.53 (dt, 1 H, J = 10.8, 4.2 Hz, H-1′),
4.49 (dd, 1 H, J = 7.2, 4.2 Hz, H-1), 4.37 (t, 1 H, J = 7.2 Hz, H-6), 4.02 (dd, 1H, J = 10.8, 7.2 Hz,
H-5), 3.87 (dd, 1H, J = 7.8, 2.4 Hz, H-3), 3.60 (dd, 1H, J = 10.8, 7.8 Hz, H-4), 2.09-2.04 (m, 1 H),
1.98-1.89 (m, 1 H), 1.70-1.63 (m, 2H), 1.48, 1.44, 1.43, 1.33 (4 s, 4 × 3 H, 2 × C(CH3)2),
1.43-1.37 (m, 1H), 1.09-0.97 (m, 2 H), 0.90 (d, 3 H, J = 6.6 Hz, -CH3), 0.88 [d, 3H, J = 7.2 Hz,
CH(CH3)2), 0.88-0.82 (m, 1H), 0.77 [d, 3H, J = 7.2 Hz, CH(CH3)2]. The data agreed well with
those reported in the literature.30

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Synthesis of (+)-1. A mixture of (+)-2 (80 mg, 150 μmol) and KOH in MeOH (3 mL, 25% w/v)
was refluxed until TLC indicated the completion of reaction. The solvent was removed in vacuum,
and the residue was suspended in saturated aq. ammonium chloride solution and extracted with
CH2Cl2. The organic phases were pooled, dried (Na2SO4), filtered, and concentrated. The residue
was purified by column chromatography to produce (+)-1 as colorless syrup (36 mg, 68%). [α] =
+19.5° (c = 0.5, CHCl3); 1H NMR (600 MHz, CDCl3): δ 7.39-7.26 (m, 5H, Ph), 4.78 (d, 1 H, J =
10.8 Hz, CH2Ph), 4.65 (d, 1 H, J = 10.8 Hz, CH2Ph), 4.45 (dd, 1 H, J = 7.2, 3.6 Hz, H-1), 4.35 (t, 1
H, J = 7.2 Hz, H-6), 4.18 (dd, 1 H, J = 10.2, 7.2 Hz, H-5), 4.05-4.01 (m, 1H, H-2), 3.92 (dd, 1 H, J
= 7.8, 2.4 Hz, H-3), 3.56 (dd, 1 H, J = 10.2, 8.4 Hz, H-4), 2.58 (d, 1 H, J = 1.2 Hz, -OH), 1.53,
1
1.44, 1.37 [3 s, 4 × 3 H, 2 × C(CH3)2]. Its H NMR spectrum was identical to that of (±)-1 and
agreed well with that of the reported.28-30
Synthesis of (+)-3. To a solution of (+)-1 (8 mg, 23 μmol) dissolved in the mixture of pyridine (0.5
mL) and DCM (0.5 mL) was added TEA (0.1 ml) followed by diphenyl chlorophosphate (18 mg,
69 μmol). The solution was stirred at rt overnight. After TLC showed the completion of reaction,
the solvents were evaporated in vacuum, and the residue was purified by column chromatography
1
(petroleum ether and ethyl acetate 3:1) to give (+)-3 as colorless syrup (10 mg, 81%). H NMR
(600 MHz, CDCl3): δ 7.35-7.13 (m, 15 H, Ph), 4.90 (td, 1 H, J = 9.6, 3.0 Hz, H-1), 4.69 (d, 1 H, J
= 12.0 Hz, CH2Ph), 4.65 (d, 1 H, J = 12.0 Hz, CH2Ph), 4.52-4.47 (m, 1 H, H-2), 4.36 (t, 1 H, J =
7.2 Hz, H-6), 4.00 (dd, J = 10.8, 7.8 Hz, 1 H, H-5), 3.95 (dd, J = 7.8, 3.0 Hz, 1 H, H-3), 3.54 (dd, J
= 10.8, 8.4 Hz, 1 H, H-4), 1.49, 1.43, 1.38, 1.32 [4 s, 4 × 3 H, 2 × C(CH3)2]. The data agreed well
with those reported in the literature.28-30
Synthesis of 1L-Ins-1-P. To a solution of (+)-3 (3 mg, 5 mol) in EtOH (1 mL) were added PtO2
(1 mg) and 3 drops of concentrated HCl. The mixture was vigorously stirred under H2 atmosphere
at rt for 2 d. The mixture was filtered, and the filtrate was evaporated in vacuum to give
1L-Ins-1-P as a white solid (1 mg, 90%), which did not need additional purification. 1H NMR (600
MHz, D2O): δ 4.20 (t, 1 H, J = 3.0 Hz, H-2), 3.80 (dt, 1 H, J = 9.6, 3.0 Hz, H-1), 3.70 (t, 1 H, J =
9.6 Hz, H-6), 3.59 (t, 1 H, J = 9.6 Hz, H-5), 3.51 (dd, 1 H, J = 9.6, 3.0 Hz, H-3), 3.28 (t, 1 H, J =

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9.6 Hz, H-4). The data agreed well with those reported in the literature.31
Synthesis of 1D-Ins-1-P. This synthesis followed exactly the same procedure described for the
synthesis of 1L-Ins-1-P, except that (+)-methyl chloroformate, instead of (-)-methyl chloroformate,
was used as the auxiliary agent for the racemic resolution. Similar reaction yields were obtained,
and the spectroscopic and other analytical data of the synthetic intermediates and final product
agreed well with those reported in the literature. 31
Acknowledgements
This research was supported by the National Basic Research (973) Program (2012CB822102) and
the National Natural Science Foundation (21472114 and 21672129) of China. We thank Mr.
Zhicheng Gu for some NMR measurements.
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Biochemical Studies of Inositol N-Acetylglucosaminyltransferase Involved in
Mycothiol Biosynthesis in Corynebacterium diphtheria
Yuchuan Guo,a,b Lizhen Wang,a Jiatong Guo,b Guofeng Gu,a,* and Zhongwu Guob,*
TOC graphic:
First-time expression, isolation, biochemical characterization, and mutagenesis studies of a MshA
from Corynebacterium diphtheria involved in its mycothiol biosynthesis