Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6

Article abstract of DOI:10.1016/j.bmcl.2014.08.057

Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-γ-lactamase activity and (-)-γ-lactamase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential γ-lactamases: a type I (+)-γ-lactamase and a (-)-γ-lactamase, making it the first strain to contain two totally different enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically pure (+)-γ-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (-)-γ-lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I (+)-γ-lactamase belongs to the amidase signature family, with 504 amino acids; the (-)-γ-lactamase, which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum USDA contains a (-)-γ-lactamase in addition to a (+)-γ-lactamase, and it is the (-)-γ-lactamase from this strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-γ-lactam with nearly 50% isolated yield and >99% ee was achieved.

Full text of DOI:10.1016/j.bmcl.2014.08.057

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4899–4902
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enzymatic preparation of optically pure
(+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (ꢀ)-
c-lactamase
from Bradyrhizobium japonicum USDA 6
,à
à
⇑
Shaozhou Zhu , Lu Ren , Songzhu Yu, Cuiyu Gong, Dawei Song, Guojun Zheng
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2014
Revised 27 July 2014
Accepted 5 August 2014
Available online 2 September 2014
Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-
mase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential
c
-lactamase activity and (ꢀ)-
-lactamases: a
-lactamase, making it the first strain to contain two totally different
enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically
pure (+)-
c-lacta-
c
type I (+)-
c
-lactamase and a (ꢀ)-
c
c
-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (ꢀ)- -lactam ((ꢀ)-2-azabi-
c
cyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various
Keywords:
carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I
c
-Lactam
(+)- -Lactamase
-Lactamase
(+)-
which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum
-lactamase in addition to a (+)- -lactamase from this
-lactam with
c
-lactamase belongs to the amidase signature family, with 504 amino acids; the (ꢀ)-
c-lactamase,
c
(ꢀ)-
c
USDA contains a (ꢀ)-
c
c
-lactamase, and it is the (ꢀ)-
c
Biocatalysis
strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-
c
Enzymatic resolution
nearly 50% isolated yield and >99% ee was achieved.
Ó 2014 Elsevier Ltd. All rights reserved.
Since the thalidomide tragedy, stricter regulations have been
introduced by the FDA on enantiomerically pure compounds. As
a consequence, chiral drugs are now almost exclusively developed
as single enantiomers.1 Recently, the enzymatic resolution of race-
mic drug intermediates has been gaining more and more attention
over the past few years due to high stereoselectivity and low cost.2
One successful example is the preparation of optically pure
microorganisms containing
far.3,4,7–14
c
-lactamase have been studied thus
c
-Lactamase is a type of enzyme that can cleave the enantiomer
-lactam (Fig. 1). This name is given according to its industrial
activity since its in vivo activity is unknown.15 Based on enantiose-
lectivity, -lactamase and
-lactamases can be divided into (ꢀ)-
(+)- -lactamase.
of
c
c
c
c
c-lactam by
c
-lactamase.3
In past research involving a rational genome mining method,
c
-Lactams such as ( )-2-azabicyclo[2.2.1]hept-5-en-3-one are
our group successfully predicted that Bradyrhizobium japonicum
important precursors to several pharmaceutically active
compounds, which were initially used for the synthesis of carbocy-
clic nucleosides.4 Later, it was found that both enantiomers are
extremely versatile synthons, and they are being used in a growing
USDA 6 may have the (+)-c
-lactamase gene.6 When we tested
the whole cell’s lactamase activity, we were surprised to discover
that the whole cell of B. japonicum USDA 6 also showed some
(ꢀ)-
c
-lactamase activity, making it the first example of a cell that
number of drug candidates.5 Different types of
c-lactamases that
contains both (+)-
c
-lactamase and (ꢀ)-
c
-lactamase.
could improve the efficiency of the preparation of these optically
pure synthons have been discovered by activity-based screening
methods and genome mining-based methods.3,6 Even so, few
A legume-root nodulating species, B. japonicum USDA 6 is a
symbiotic nitrogen-fixing bacterium. Under laboratory conditions,
we found that it is a weak biocatalyst for industrial application. The
strain grows very slowly, and because the whole cell contains both
(+)-
c
-lactamase and (ꢀ)-
c-lactamase, the stereoselectivity of the
⇑
Corresponding author. Tel./fax: +86 10 64437507.
whole cell is poor. Therefore, heterologous expression of these
enzymes in another host is needed.
We have already heterologously expressed (+)-
Escherichia coli and examined it in detail in a previous study; the
E-mail address: zhenggj@mail.buct.edu.cn (G. Zheng).
Current address: Department of Chemistry, Philipps-University Marburg,
Hans-Meerwein-Strasse 4 and LOEWE-Centre for Synthetic Microbiology, D-35032
Marburg, Germany.
c-lactamase in
à
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2014.08.057
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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S. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4899–4902
Figure 1. Enzymatic preparation of optically pure c-lactams as synthetic building blocks in the synthesis of various carbocyclic nucleoside drugs.
results showed that the recombinant (+)-
504 amino acids, could only specifically cleave the enantiomer of
(+)-
-lactam.6 To demonstrate that the strain also contains
(ꢀ)- -lactamase, the cloning, purification, and characterization of
the (ꢀ)- -lactamase from B. japonicum USDA 6 are reported in
this Letter. Preparation of both optically pure (+)- -lactam and
(ꢀ)- -lactam by these two enzymes is also described here.
The gene of (ꢀ)- -lactamase (BJ6T_13230) from B. japonicum
c-lactamase, which has
c
c
c
c
c
c
USDA 6, which has 274 amino acids, was constructed in the vector
pET28a(+) and expressed in E. coli BL 21.16 A single colony was
incubated at 37 °C in an orbital shaker (220 rpm) for about 24 h.
2 mL of an overnight culture of E. coli BL 21 (DE3) carrying the
desired expression construct was diluted into a 500 mL baffled
flask containing 200 mL of fresh LB medium supplemented with
50 lg/mL kanamycin. When the OD reached 0.6, lactose was added
to a final concentration of 5 g/L. After incubation for another 6 h,
cells were harvested by centrifugation (6000g) for 10 min and sus-
pended in phosphate buffer, and then sonicated for the subsequent
purification. Purification of the fusion protein on the Ni-chelating
column was carried out using the protocol and buffer supplied
by GE Novagen.
After purification, a single band was observed on the SDS-PAGE
gel (Fig. 2), and the physical properties of the enzyme were inves-
tigated. One unit of (ꢀ)-
enzyme required to hydrolyze 1
the effect of the temperature on (ꢀ)-
from 20 to 70 °C for 12 h by adding 50
(0.772 mg/ml) to 500 l of the 4.6 mM substrate solution (pH 7.5
c
-lactamase is defined as the amount of
mol of substrate in 12 h.17 First,
-lactamase was measured
pure enzyme
l
c
ll
l
Tris–HCl buffer), and the highest activity was set to 100%. The
results show that the optimum temperature was 50 °C. When the
reaction temperature was above 60 °C, the activity decreased
rapidly, and the enzyme was inactivated at 70 °C (Fig. 3A). In order
to examine the thermostability of the enzyme, the enzyme was
incubated in the assay buffer without substrate at 20–70 °C for
12 h, and then the reaction was conducted by adding the substrate
at 50 °C for 12 h. The results illustrate that the enzyme showed
good stability below 50 °C. However, at temperatures above
60 °C, only 5% activity remained, indicating that the enzyme could
not withstand high temperatures (Fig. 3B).
Figure 2. Coomassie-stained gel after SDS–PAGE of purified and concentrated
histidine-tagged (ꢀ)- -lactamase.
c
Next, the optimum reaction pH was studied at 50 °C. The reac-
tion was carried out at pH 4.0–10.0 with the same reaction mixture
as before. We found that the optimum pH of the enzyme was 6.0;
at the extreme pH values of 4.0 and 10.0, almost all activity was
lost (Fig. 3C). The stability of the pH was also examined. The
enzyme was incubated at 30 °C in the buffer (pH 4.0–10.0) for
12 h, and then residual activity was measured under the standard
conditions with the same amount of enzyme as before. The activity
of the enzyme without treatment was set to 100%, and the residual
activity was measured. To our surprise, the enzyme showed high
pH stability both in the neutral and in the alkaline conditions, with
over 90% activity remaining after the reaction (Fig. 3D).
The effects of metal ions (Ca2+, Cu2+, Fe3+, Fe2+, Zn2+, Mn2+, K+,
Ni2+, Li+, and Na+) were examined at the final concentration of
1 mM. The reaction was carried out under the standard conditions,
and the activity of the enzyme without metal ions added into the
mixture was measured as a control group and set to 100%. We
could see that the enzyme maintained its activity with the addition

S. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4899–4902
4901
Figure 3. Physical properties of (ꢀ)-
c
-lactamase: (A) effect of temperature; (B) effect of temperature on stability; (C) effect of pH; and (D) effect of pH on stability.
transformation of velocity data obtained by varying the initial sub-
strate concentrations showed a good linear regression fit (R2 >0.99
for all plots). From the plots, Km and Vmax values were determined;
the Km of the enzyme was 2.4 ꢁ 10ꢀ3
lM, and the Vmax was
lM/s. The Km data indicate that the enzyme had a great
1.3 ꢁ 10ꢀ3
affinity for the substrate.
After we optimized the reaction condition, a small-scale exper-
iment was conducted under the optimized conditions. As Figure 5
shows, the optical purity of the chiral product reached over 99%
enantiomeric excess through hydrolysis of the (racemic)
by (ꢀ)- -lactamase; compared to the reaction by the whole cell,
the enantioselectivity was greatly improved. And while the recom-
binant (+)- -lactamase could only cleave the (+)- -lactam, the
recombinant (ꢀ)- -lactamase from the same strain could specifi-
cally hydrolyze the other enantiomer of -lactam. This demon-
c-lactam
c
c
c
c
c
strates that two totally different enantioselective lactamases
were actually involved in the degradation of the lactam when we
used the whole cell as a biocatalyst.
Figure 4. The effects of various metal ions on the activity of (ꢀ)-
c-lactamase.
In summary, we have demonstrated that B. japonicum USDA 6
contains a (ꢀ)-
is the first example of a single strain containing two opposite chi-
rally selective -lactamases. By heterologous expression in E. coli,
we greatly improved the enantioselectivity compared to the
whole-cell catalyst. The recombinant enzymes could easily be
c-lactamase in addition to a (+)-c-lactamase. This
of most metal ions (Fig. 4). No ion could strongly enhanced the
activity, and this suggested that it was not a metalloenzyme.
However, only 50% activity remained when Fe2+ was present, and
near inactivation was observed under Cu2+, Fe3+, and Zn2+. These
heavy metals probably could disrupt the secondary and tertiary
structure of the enzyme and lead to denaturation.
c
applied in the preparation of either optically pure (+)-
optically pure (ꢀ)- -lactam. Both enzymes catalyzed efficient res-
olution, resulting in optically pure -lactam with nearly 50% iso-
lated yield and >99% ee. These versatile -lactamases will enrich
researchers’ enzyme toolkits and will play an increasingly
c-lactam or
c
The kinetic parameters were determined by Lineweaver–Burk
c
plot. Initial substrate concentrations were varied from
3 to
c
10 mM. In order to make sure the measurements remained in the
linear range, the reaction was stopped at 1, 2, 3, 4, and 5 h. Linear

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S. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4899–4902
Figure 5. Chiral-HPLC analysis of resolution of racemic c-lactam by pure (+)-c-lactamase, (ꢀ)-c-lactamase and the whole cell of B. japonicum USDA 6.
9. Toogood, H. S.; Brown, R. C.; Line, K.; Keene, P. A.; Taylor, S. J. C.; McCague, R.;
important role as biocatalysts in the synthesis of key intermediates
for the chemical and pharmaceutical industries.
Littlechild, J. A. Tetrahedron 2004, 60, 711.
10. Wang, J.; Zhang, X.; Min, C.; Wu, S.; Zheng, G. Process Biochem. 2011, 46, 81.
11. Line, K.; Isupov, M. N.; Littlechild, J. A. J. Mol. Biol. 2004, 338, 519.
12. Song, D.; Zhu, S.; Li, X.; Zheng, G. J. Mol. Graph. Model. 2014, 47, 1.
13. Torres, L. L.; Schliessmann, A.; Schmidt, M.; Silva-Martin, N.; Hermoso, J. A.;
Berenguer, J.; Bornscheuer, U. T.; Hidalgo, A. Org. Biomol. Chem. 2012, 10, 3388.
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16. The (ꢀ)-
c-lactamase gene were amplified by PCR. The primers used were: The
forward primer LactFP was 50-ggaattccatatgcccaccatcaccacc-30 and the reverse
primer LactRP was 50-cccaagctttcacgccttgaagaacgcc-30 the NdeI and HindIII
sites were added to the primers, respectively.
17. HPLC analysis: sample were analyzed by HPLC on a Shimadzu HPLC (interfaced
with Shimadzu LCsolution Software) equipped with a detector at 260 nm and a
Daicel Chiralpak AS-H column (Daicel Corp., Tokyo, Japan) and eluted with a
mobile phase consisting of 90% acetonitrile and 10% isopropanol (volume ratio)
at a flow rate of 0.6 ml/min.
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