57NISR Research GRANT
2008 Young Investigator Research Grant

Molecular breeding of enzymes catalyzing asymmetric reactions: Aimed to provide a non-natural amino acid chemical library
Hisashi MURAMATSU
Kochi University, Research and Education Faculty, Natural Sciences Cluster, Agriculture Unit

Research aims

Results

Nonproteinous amino acids, which are not the building
blocks of protein, are useful for the building blocks in medicines1–4). However, many optically active nonproteinous
amino acids are expensive. Therefore, development of alow-cost and practical asymmetric-reaction-process of nonproteinous amino acids is expected. In this study, we aimed
to improve thermostability and substrate specificity of thetype II malate/lactate dehydrogenase family protein, DpkA
which is useful for asymmetric synthesis by evolutionary
molecular engineering. DpkA catalyzes the NADPH-dependent formation of N-methyl-L-amino acids from a -ketoacids and methylamine and the NADPH-dependent asymmetric reduction of D1-piperideine-2-carboxylate to form Lpipecolate5,6). Hence, DpkA is available for production ofthese optically active compounds, which are useful asbuilding blocks in medicines and pesticides. We consider
that the improved DpkA will contribute to the developmentof a practical and sustainable asymmetric-reaction-process.

1)

Methods
A dpkA (PP3591) gene from Pseudomonas putida
NBRC100650 (KT2440) was ligated into the NdeI-HindIIIsite of pET21a(). The resultant plasmid was named
pDpkA. Recombinant Escherichia coli BL21(DE3) cells
harboring pDpkA were grown in LB medium containing
100 m g/ml ampicillin. After the induction of gene expression with 1 mM IPTG, the cells were harvested by centrifugation, disrupted by sonication on ice and centrifuged. TheC-terminal six-histidine tagged DpkA was purified from theresultant crude extract by Ni-nitrilotriacetic acid column
chromatography. Stabilities of DpkA against temperature,pH, NaCl, guanidine hydrochloride, SDS and ethanol were
determined using the purified DpkA. Next, the optimal condition of error-prone PCR was examined to construct a mutant enzyme (DpkA) library. Moreover, a selection method
for thermostabilized enzyme from the mutant enzyme library was examined.

Stability of DpkA.
The stability of DpkA under various conditions is shownin Fig. 1. After incubation at 25oC for 30 min, DpkA did
not retain its activity. The enzyme showed 59% activity
after incubation at 30oC for 30 min and was inactivated byincubation at 45oC for 2.5 min (Fig. 1, A). Above pH 7, theactivity was decreased as pH increased (Fig. 1, B). DpkA
showed 63% activity in the reaction mixture containing
0.85% NaCl (Fig. 1, C). The enzyme showed 24% activity
by treatment with 13 m M guanidine hydrochloride (Fig. 1,D). DpkA was inactivated by treatment with 20 m M SDSand 60% ethanol (Fig. 1, E and F).
2) Optimization of the conditions for error-prone PCR
and selection method for thermostabilized enzyme from themutant library.
Plasmid pDpkA was used as a template in mutagenesis
for error-prone PCR. A 100 m l reaction mixture contained
10 m l 10PCR buffer, 0.25 mM dGTP, 0.25 mM dCTP, 0.25mM dTTP, 4 m M dATP, 33.5 pmol primers, 3.4 ng template
DNA and 4.2 U of Taq polymerase (Takara). PCR was performed with an automatic thermal cycler for 10 cycles consisting of 94oC for 1 min, 55oC for 1 min and 72oC for 1min, followed by 25 cycles under the same conditions afterthe addition of 25 nmol of ATP only. Amplified mutantdpkA DNA fragments were ligated into pET21a() and E.
coli BL21(DE3) was transformed by the resultant plasmids.
Plasmid DNA harboring mutant dpkA was extracted fromtransformants and sequenced. Each extracted plasmid hadfrom one to six substituted bases within the inserted genes.
Therefore, we decided to prepare mutant dpkA by this PCR
conditions in this study.
Next, the selection method for thermostabilized enzyme
from the mutant library was examined. Crude extract fromE. coli BL21(DE3) harboring pDpkA was incubated at 30,45, and 50oC for 30 min. After incubation, N-methyl-Lamino acid dehydrogenase activity in the crude extract wasassayed by a microplate reader. Crude extracts incubated at
30 and 45oC showed activity but crude extract incubated at50oC was inert. Therefore, DpkA in the crude extract wasdenatured at 50oC. From these result we decided to selectthermostabilized DpkA by comparing the activities of wild-58

Fig. 1. Stability of DpkA. Effects of temperature (A), pH (B), concentration of NaCl (C), concentration of guanidine hydrochloride (D), concentration ofSDS (E) and concentration of ethanol (F) on the stability of DpkA.
DpkA was incubated on ice for 30 min in experiments shown in (B) or for 10 min in experiments shown in (D), (E) and (F).

type DpkA incubated at 45oC and 50oC with those of mutant DpkAs incubated at the same temperatures.
We will attempt to obtain thermostabilized DpkA by theabove-mentioned method. Moreover, we aim to obtain not
only thermostabilized DpkA but also NADH-dependent
DpkA by error-prone PCR.

DpkA from the mutant enzyme library. We will attempt tosynthesize chiral building blocks in medicines and pesticides such a N-methyl-L-amino acids and L-pipecolate bymodified DpkA. The reaction process is expected to makelong-term asymmetric synthesis by the enzyme possible
and to increase productivity.

Conclusion

References

This work demonstrated the stability of DpkA from P.
putida NBRC100650 under various conditions. Moreover,
we could construct a mutant enzyme library by optimized
error-prone PCR. The selection method for thermostabilized enzyme from the library was also determined. Hereafter, we aim to obtain thermostable or NADH-dependent

1)

2)

Rinehart K. L. Jr., Gloer J. B., Cook J. C. Jr., Mizsak S. A., Scahill T.
A. Structures of the didemnins, antiviral and cytotoxic depsipeptides
from a Caribbean tunicate. J. Am. Chem. Soc. 103: 1857–1859(1981).
Pettit G. R., Kamano Y., Herald C. L., Fujii Y., Kizu H., Boyd M.
R., Boettner F. E., Doubek D. L., Schmidt J. M., Chapuis J.-C.,
Michel C. Isolation of dolastatins 10–15 from the marine mollusc

593)

4)

5)

Dolabella auricularia. Tetrahedron 49: 9151–9170 (1993).
Tanaka H., Kuroda A., Marusawa H., Hatanaka H., Kino T., Goto T.,
Hashimoto M., Taga T. Structure of FK506: a novel immunosuppressant isolated from Streptomyces. J. Am. Chem. Soc. 109: 5031–5033(1987).
Germann U. A., Shlyakhter D., Mason V. S., Zelle R. E., Duffy J. P.,
Galullo V., Armistead D. M., Saunders J. O., Boger J., Harding M. W.
Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance invitro. Anticancer Drugs 8: 125–140 (1997).
Muramatsu H., Mihara H., Kakutani R., Yasuda M., Ueda M., Kuri-6)

hara T., Esaki N. The putative malate/lactate dehydrogenase from
Pseudomonas putida is an NADPH-dependent D 1-piperideine-2-carboxylate/D 1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline. J. Biol. Chem. 280: 5329–5335(2005).
Mihara H, Muramatsu H, Kakutani R, Yasuda M, Ueda M, KuriharaT, Esaki N. N-methyl-L-amino acid dehydrogenase from
Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily. FEBS J. 272: 1117–1123(2005).