Enzymes responsible for the conversion of Nα-[(benzyloxy) carbonyl]-D-lysine to Nα-[(benzyloxy)carbonyl]-D-aminoadipic acid by Rhodococcus sp. AIU Z-35-1

Article abstract of DOI:10.1002/cbdv.200900251

The enzymes responsible for the conversion of N^α- [(benzyloxy)carbonyl]-D-lysine (N^α-Z-D-lysine) to N ^α-Z-D-aminoadipic acid (N^α-Z-D-AAA) by Rhodococcus sp. AIU Z-35-1 were identified. N^α-Z-D-Lysine was first converted to N^α-Z-D-aminoadipic δ-semialdehyde (N^α-Z-D-AASA) by D-specific amino acid deaminase, whereas N^α-Z-L-lysine was converted to N^α-Z-L-AASA by L-specific amino acid oxidase. The resulting N^α-Z-D-AASA was then converted to N^α-Z-D-AAA by the same aldehyde dehydrogenase that is responsible for N^α-Z-L-AASA oxidation. The product amount of the D-specific amino acid deaminase reached the maximum at one day of cultivation in the L-lysine medium. The aldehyde dehydrogenase reached the maximum at three days of cultivation.

Full text of DOI:10.1002/cbdv.200900251

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Enzymes Responsible for the Conversion of Na-[(Benzyloxy)carbonyl]-d-
lysine to Na-[(Benzyloxy)carbonyl]-d-aminoadipic Acid by Rhodococcus sp.
AIU Z-35-1
by Kimiyasu Isobe*, Nahoko Fukuda, Shouko Nagasawa, and Kaoru Saitou
Department of Biological Chemistry and Food Science, Iwate University, 18-8 Ueda-3,
Morioka 020-8550, Japan (phone/fax: þ81-19-621-6155; e-mail: kiso@iwate-u.ac.jp)
The enzymes responsible for the conversion of Na-[(benzyloxy)carbonyl]-d-lysine (Na-Z-d-lysine)
to Na-Z-d-aminoadipic acid (Na-Z-d-AAA) by Rhodococcus sp. AIU Z-35-1 were identified. Na-Z-d-
Lysine was first converted to Na-Z-d-aminoadipic d-semialdehyde (Na-Z-d-AASA) by d-specific amino
acid deaminase, whereas Na-Z-l-lysine was converted to Na-Z-l-AASA by l-specific amino acid oxidase.
The resulting Na-Z-d-AASA was then converted to Na-Z-d-AAA by the same aldehyde dehydrogenase
that is responsible for Na-Z-l-AASA oxidation. The product amount of the d-specific amino acid
deaminase reached the maximum at one day of cultivation in the l-lysine medium. The aldehyde
dehydrogenase reached the maximum at three days of cultivation.
Introduction. – l-a-Aminoadipic d-semialdehyde (l-a-AASA) and l-a-amino-
adipic acid (l-a-AAA) are precursors of biosynthesis of b-lactam antibiotics, and both
compounds and their derivatives provide interesting raw materials for the chemical
synthesis of pharmaceuticals or physiologically active peptides. Some biochemical
methods for the production of l-a-AASA, l-a-AAA, and their related compounds
have been developed using l-lysine-6-aminotransferase or l-lysine-6-dehydrogenase
[1][2]. However, these methods had some drawbacks such as the requirement of a
second substrate or cofactor regeneration system, and the yields were low. We,
therefore, isolated a new bacterial strain, Rhodococcus sp. AIU Z-35-1, as a high
producer of Na-Z-l-AAA from Na-Z-l-lysine, to overcome the above drawbacks, and
developed an efficient method for production of Na-Z-l-AAA by the cell reaction [3].
This strain was also useful for the production of Na-Z-l-AASA, Na-Z-d-AASA, and
Na-Z-d-AAA, since Na-Z-l-lysine was converted to Na-Z-l-AAA via Na-Z-l-AASA
and Na-Z-d-lysine was converted to Na-Z-d-AAA via Na-Z-d-AASA [3][4]. Recently,
we identified the enzymes catalyzing the conversion of Na-Z-l-lysine to Na-Z-l-AAA
[5][6]. However, the enzymes responsible for the conversion of Na-Z-d-lysine to Na-Z-
d-AAA have not been identified. Therefore, the present article describes the
identification of the enzymes catalyzing the conversion of Na-Z-d-lysine to Na-Z-d-
AAA. The optimal cultivation time for the production of these enzymes was also
determined.
Results and Discussion. – Identification of the Enzyme Catalyzing the Conversion of
Na-Z-d-Lysine to Na-Z-d-AASA. Cells from Rhodococcus sp. AIU Z-35-1, harvested
from the l-lysine medium after 1 d of cultivation, were incubated with Na-Z-d-lysine,
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
and the reaction products were analyzed. In this reaction, formation of Na-Z-d-AASA
and NH3, but not of H2O2, was observed (data not shown). When the crude enzyme
solution was incubated with Na-Z-d-lysine, formation of Na-Z-d-AASA and NH3 was
also detected, but that of H2O2 and NAD(P)H was not (data not shown). In addition,
the produced amount of Na-Z-d-AASA increased in parallel with that of NH3 (Fig. 1).
Then, the stereospecificity of the enzyme was analyzed using the partially purified
enzyme. The formation of NH3 was detected from Na-Z-d-lysine, but not from Na-Z-l-
lysine. These results indicate that the conversion of Na-Z-d-lysine to Na-Z-d-AASA
was catalyzed by a d-specific amino acid deaminase in Rhodococcus sp. AIU Z-35-1,
according to Scheme 1.
Fig. 1. Formation of NH3 and Na-Z-d-AASA from Na-Z-d-lysine. Na-Z-d-Lysine (30 mmol) was
incubated with 0.009 units of d-amino acid deaminase at 308 and pH 7.0. Open circles, Na-Z-d-AASA;
closed circles, NH3.
Scheme 1. Conversion of Na-Z-d-Lysine to Na-Z-d-AASA by Rhodococcus sp. AIU Z-35-1
We have already reported [5] that when Na-Z-l-lysine was used as substrate, its
conversion to Na-Z-l-AASA was catalyzed by an oxidase purified from Rhodococcus
sp. AIU Z-35-1 and that the amount of H2O2 produced increased in parallel with that of
NH3 and Na-Z-l-AASA. We further revealed that this enzyme also exhibited oxidase
activity on Na-acetyl-l-lysine, Ne-acetyl-l-lysine, and l-amino acids, except for glycine,
l-proline, and l-cysteine, but not on Na-Z-d-lysine, Na-acetyl-d-lysine, Ne-acetyl-d-
lysine, and d-amino acids [5]. Thus, Na-Z-l-lysine was converted to Na-Z-l-AASA by
the l-specific amino acid oxidase with broad substrate specificity in Rhodococcus sp.
AIU Z-35-1.

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In our studies on the enzymes responsible of the conversion of Na-Z-d-lysine to Na-
Z-d-AASA and Na-Z-l-lysine to Na-Z-l-AASA, it was concluded that Na-Z-d-lysine
was converted to Na-Z-d-AASA by the d-specific amino acid deaminase, whereas Na-
Z-l-lysine was converted to Na-Z-l-AASA by the l-specific amino acid oxidase in
Rhodococcus sp. AIU Z-35-1. We have already revealed in an earlier report [7] that Na-
Z-l-lysine and Na-Z-d-lysine were converted to Na-Z-l-AASA and Na-Z-d-AASA,
respectively, by amine oxidase from Aspergillus niger AKU 3302. Thus, the enzyme
catalyzing the conversion of Na-Z-d-lysine to Na-Z-d-AASA in the Rhodococcus strain
was also different from that of A. niger.
Identification of the Enzyme Catalyzing the Conversion of Na-Z-d-AASA to Na-Z-
d-AAA. When the crude enzyme solution, which was prepared from the cells incubated
in the l-lysine medium, was incubated with Na-Z-d-AASA, no formation of H2O2 and
Na-Z-d-AAA was detected. However, formation of Na-Z-d-AAA was observed when
NADþ was added to the solution. Moreover, the produced amount of NADH increased
in parallel with that of Na-Z-d-AAA (Fig. 2). Thus, it was concluded that Na-Z-d-
AASA was converted to Na-Z-d-AAA by aldehyde dehydrogenase, according to
Scheme 2. Then, the substrate specificity of this aldehyde dehydrogenase was analyzed
with the purified enzyme. This enzyme also exhibited dehydrogenase activity towards
Na-Z-l-AASA. These results indicated that the enzyme catalyzing the conversion of
Fig. 2. Formation of NADH and Na-Z-d-AAA from Na-Z-d-AASA. Na-Z-d-AASA (30 mmol) was
incubated with 0.02 units of aldehyde dehydrogenase at 308 and pH 7.0. Open circles, Na-Z-d-AAA;
closed circles, NADH.
Scheme 2. Conversion of Na-Z-d-AASA to Na-Z-d-AAA by Rhodococcus sp. AIU Z-35-1

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Na-Z-d-AASA to Na-Z-d-AAA did not show stereospecificity, and both conversions
Na-Z-d-AASA to Na-Z-d-AAA and Na-Z-l-AASA to Na-Z-DLAAA were catalyzed
by the same aldehyde dehydrogenase in Rhodococcus sp. AIU Z-35-1.
Production of Enzyme. Rhodococcus sp. AIU Z-35-1 was incubated in the l-lysine
medium at 308 for four days, and the Na-Z-d-lysine deaminase activity and the Na-Z-d-
AASA dehydrogenase activity were assayed each day using cell-free extract. The Na-Z-
d-lysine deaminase activity reached the maximum at 1 d of cultivation and was reduced
by a longer cultivation time. In contrast, the Na-Z-d-AASA dehydrogenase activity
reached the maximum at three days of cultivation (Fig. 3). These results were in good
agreement with our previous results, which indicated that a selective production of Na-
Z-d-AASA was achieved using the cells harvested after one day of cultivation in the l-
lysine medium and that Na-Z-d-AAAwas efficiently produced using the cells harvested
after two days of cultivation.
Fig. 3. Production of d-amino acid deaminase and aldehyde dehydrogenase. Rhodococcus sp. AIU Z-35-
1 was incubated in the l-lysine medium at 308 for 4 d, and the d-amino acid deaminase and the aldehyde
dehydrogenase activities were assayed each day using cell-free extract. Closed circles, d-amino acid
deaminase; open circles, aldehyde dehydrogenase; triangles, cell growth (OD660 nm).
Conclusions. – To identify the enzymes catalyzing the conversion of Na-Z-d-lysine
to Na-Z-d-AAA in Rhodococcus sp. AIU Z-35-1, a crude enzyme solution from the
cells cultivated in the l-lysine medium were prepared, and the enzyme activities
towards Na-Z-d-lysine and Na-Z-d-AASAwere assayed. When Na-Z-d-lysine was used
as substrate, formation of NH3, but not of H2O2 and NAD(P)H, was observed. In
addition, the product amounts of NH3 and Na-Z-d-AASA were stoichiometrically
increased by the reaction with partially purified enzyme. This enzyme did not catalyze
the deamination of Na-Z-l-lysine. It was, therefore, concluded that the conversion of
Na-Z-d-lysine to Na-Z-d-AASA was catalyzed by d-specific amino acid deaminase in
Rhodococcus sp. AIU Z-35-1.
When Na-Z-d-AASA was incubated with the crude enzyme solution, formation of
NADH and Na-Z-d-AAA, but not of H2O2, was observed. This enzyme also exhibited
dehydrogenase activity towards Na-Z-l-AASA. It was therefore concluded that Na-Z-

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d-AASA was converted to Na-Z-d-AAA by non-stereospecific aldehyde dehydrogen-
ase.
In our previous studies of the enzymes responsible for the production of Na-Z-l-
AASA and Na-Z-l-AAA from Na-Z-l-lysine by the cells of Rhodococcus, we have
revealed that Na-Z-l-AASAwas produced from Na-Z-l-lysine by l-specific amino acid
oxidase, and the resulting Na-Z-l-AASA was converted to Na-Z-l-AAA by aldehyde
dehydrogenase. It was therefore concluded that Na-Z-l-AAA and Na-Z-d-AAA were
produced from Na-Z-l-lysine and Na-Z-d-lysine by the combination of l-amino acid
oxidase and non-stereospecific aldehyde dehydrogenase, and by d-amino acid
deaminase and non-stereospecific aldehyde dehydrogenase, respectively, in Rhodo-
coccus sp. AIU Z-35-1.
Experimental Part
Chemicals. Na-[(Benzyloxy)carbonyl]-d-lysine (Na-Z-d-lysine) and Na-Z-l-lysine were purchased
from Watanabe Chemical Industries (Hiroshima, Japan), and b-NADþ and NADPþ were obtained from
Oriental Yeast (Osaka, Japan). Na-Z-d-aminoadipic d-semialdehyde (Na-Z-d-AASA) and Na-Z-l-
AASA were prepared according to the method described in [4]. Peroxidase was a gift from Amano
Enzyme (Nagoya, Japan). All other chemicals used were of analytical grade and commercially available.
Cultivation of Microorganisms. Rhodoccocus sp. AIU Z-35-1 was incubated in an l-lysine medium at
308 for 1 or 2 d with shaking at 120 strokes per min, according to the method of Isobe et al. [3]. The cells
were harvested by centrifugation at 20,000g for 10 min, washed with 0.1m potassium phosphate buffer
pH 7.0, and stored at ꢀ208 until use.
Purification of Enzymes. The cells cultivated in the l-lysine medium were suspended in 1.0 ml of
10 mm buffer (pH 7.0) and disrupted with glass beads using a multi-bead shocker (Yasui Kikai, Osaka) at
2500 rpm for 8 min (4ꢁ2 min). The supernatant was obtained by centrifugation at 20,000g for 10 min and
used as crude enzyme soln. The crude enzyme soln. was applied to column chromatography (CC; DEAD-
Gigacap Q (Tosoh, Tokyo, Japan) and hydroxyapatite). The eluate from the hydroxyapatite column was
used as a partially purified d-amino acid deaminase.
Aldehyde dehydrogenase was purified from the crude enzyme soln. by CC (DEAE-Toyopearl,
Phenyl-Toyopearl, and Blue-Sepharose), according to the method of Isobe et al. [6].
Analysis of Reaction Products. The reaction products were identified by HPLC using a TSK-Gel
DEAE-5PW column (Tosoh, Tokyo, Japan), according to the method of Isobe et al. [8]. The product was
collected by monitoring the absorbance at 210 nm, and the molecular mass was then analyzed by a
Finnigan Mass Spectrometer LCQ Deca (Thermo Electron, Yokohama, Japan). The aldehyde group of
the product was confirmed using 3-methyl-2-benzothiazolinone hydrazone, according to the method of
Paz et al. [9]. The amounts of reaction products were calculated from the peak area of the HPLC
chromatograms obtained with the TSK-Gel DEAE-5PW column under the same conditions as used for
the identification of the reaction products.
Assay of Enzyme Activity. The oxidase activity was assayed by measuring the rate of H2O2 formation
as follows. The standard mixture contained 40 mmol of substrate, 0.6 mmol of 4-aminoantipyrine,
1.94 mmol of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt dihydrate, 6.7 units of
peroxidase, 0.1 mmol of potassium phosphate (pH 7.0), and an appropriate amount of enzyme in a final
volume of 1.0 ml. The color development by H2O2 formation was spectrophotometrically followed at 308
by measuring the absorbance at 555 nm [5].
The dehydrogenase activity was spectrophotometrically assayed by measuring the rate of NADH
formation as follows. The standard mixture contained 20 mmol of substrate, 0.6 mmol of b-NADþ,
0.2 mmol of potassium phosphate (pH 8.0), and an appropriate amount of enzyme in a final volume of
1.0 ml. The formation of NADH was followed at 308 by measuring the absorbance at 340 nm [6].
The deaminase activity was assayed by measuring the rate of NH3 formation by coupling with
glutamate dehydrogenase (EC 1.4.1.4, GLDH) as follows. The standard mixture contained 30 mmol of

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Na-Z-d-lysine, 0.36 mmol of b-NADPH, 20 units of GLDH, 3 mmol of 2-oxoglutaric acid, 0.1 mmol of
potassium phosphate (pH 7.5), and an appropriate amount of enzyme in a final volume of 1.0 ml. The
amount of NADPþ generated from NH3 was spectrophotometrically followed at 308 by measuring the
absorbance at 340 nm.
One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one
mmol of H2O2, NADH, or NADPH per min, resp.
REFERENCES
[1] T. Hammer, R. Bode, Zentralbl. Mikrobiol. 1992, 147, 65.
[2] T. Fujii, T. Narita, H. Agematu, N. Agata, K. Isshiki, J. Biochem. 2000, 128, 391.
[3] K. Isobe, K. Tokuta, Y. Narita, A. Matsuura, T. Sakaguchi, N. Wakao, J. Mol. Catal. B: Enzym. 2004,
32, 27.
[4] K. Isobe, S. Nagasawa, K. Tokuta, A. Matsuura, T. Sakaguchi, N. Wakao, J. Biosci. Bioeng. 2005, 100,
288.
[5] K. Isobe, S. Nagasawa, J. Biosci. Bioeng. 2007, 104, 218.
[6] K. Isobe, N. Fukuda, S. Nagasawa, J. Biosci. Bioeng. 2007, 104, 398.
[7] K. Isobe, K. Tokuta, Y. Narita, A. Matsuura, T. Sakaguchi, N. Wakao, J. Mol. Catal. B: Enzym. 2004,
30, 119.
[8] K. Isobe, K. Tokuta, Y. Narita, A. Matsuura, T. Sakaguchi, N. Wakao, J. Mol. Catal. B: Enzym. 2004,
30, 13.
[9] M. A. Paz, O. O. Blumenfeld, M. Rojkind, E. Henson, C. Furfine, P. M. Gallop, Arch. Biochem.
Biophys. 1965, 109, 548.