Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine.

Article abstract of DOI:10.1271/bbb.66.934

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.

Full text of DOI:10.1271/bbb.66.934

This article was downloaded by: [Universitat Politècnica de València]
On: 25 November 2014, At: 00:59
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Biosynthesis of Vitamin B in Rhizobium: In Vitro
6
Synthesis of Pyridoxine from 1-Deoxy-D-xylulose and
4
-Hydroxy-L-threonine
a a
a
Masaaki TAZOE , Keiko ICHIKAWA & Tatsuo HOSHINO
a
Department of Applied Microbiology, Nippon Roche Research CenterKamakura,
Kanagawa 247-8530, Japan
Published online: 22 May 2014.
To cite this article: Masaaki TAZOE, Keiko ICHIKAWA & Tatsuo HOSHINO (2002) Biosynthesis of Vitamin B in Rhizobium:
6
In Vitro Synthesis of Pyridoxine from 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine, Bioscience, Biotechnology, and
Biochemistry, 66:4, 934-936, DOI: 10.1271/bbb.66.934
To link to this article: http://dx.doi.org/10.1271/bbb.66.934
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 66 (4), 934–936, 2002
Preliminary Communication
Biosynthesis of Vitamin B in Rhizobium
:
L
In Vitro Synthesis of Pyridoxine
-threonine
6
from 1-Deoxy-
D
-xylulose and 4-Hydroxy-
Masaaki TAZOE,† Keiko ICHIKAWA, and Tatsuo HOSHINO
Department of Applied Microbiology, Nippon Roche Research Center, Kamakura,
Kanagawa 247-8530, Japan
Received December 24, 2001; Accepted December 25, 2001
Pyridoxine (vitamin B
6
) in Rhizobium is synthesized
-threonine. To
of two groups reported that pyridoxine 5
(PNP) was synthesized in vitro from 1-deoxy-
lose 5-phosphate (1-DXP) and 4-(phosphohydroxy)-
?
-phosphate
from 1-deoxy- -xylulose and 4-hydroxy-
D
L
D
-xylu-
deˆne the pathway enzymatically, we established an en-
zyme reaction system with a crude enzyme solution of
R. meliloti IFO14782. The enzyme reaction system
L
-threonine (4-PHT) as substrates in the presence of
+
only one coenzyme, NAD , by two puriˆed recom-
＋
＋
5)
required NAD , NADP , and ATP as coenzymes, and
binant proteins, PdxA (4-PHT dehydrogenase) and
6
,7)
diŠered from the E. coli enzyme reaction system
PdxJ (PNP synthase)
of E. coli. We previously
comprising PdxA and PdxJ proteins, which requires
reported that R. meliloti IFO 14782 was a PN over-
＋
8)
only NAD for formation of pyridoxine 5
?
-phosphate
-xylulose 5-phosphate and 4-(phospho-
-threonine.
producer and that PN (3) was formed by ring
from 1-deoxy-
D
closure of two precursors, 1-DX (1) and 4-HT (2),
hydroxy)-
L
which entered the C
5
(C-2
?
, 2, 3, 4 and 4
?) and NC
3
(
N-1, C-6, 5 and 5
?) units, respectively, of the PN
9
)
Key words: vitamin B
-deoxy-
6
in vitro synthesis; Rhizobium
;
molecule (Fig. 1). To identify the enzymes responsi-
ble for the pathway leading to PN from 1-DX and
4-HT in Rhizobium, we established an in vitro system
of PN synthesis from the two precursors. Here, we
present the ˆrst evidence that a crude enzyme solu-
tion prepared from a cell-free extract of R. meliloti
IFO 14782 catalyzed synthesis of PN (3) from 1-DX
(1) and 4-HT (2) in the presence of three coenzymes,
1
D
-xylulose; 4-hydroxy- -threo-
L
nine; pyridoxine
The biosynthetic study of vitamin B has been done
6
intensively in Escherichia coli from the '70s. In the
past ˆfteen years, rapid progress was made both
1
)
through the genetic investigation of Winkler et al
.
+
+
and through the tracer investigations of Spenser et
NAD , NADP , and ATP, and that the coenzymes
required were diŠerent from those of the E. coli en-
zyme reaction system containing PdxA and PdxJ,
2
–4)
al
pyridoxine (PN) was synthesized from two com-
pounds, 1-deoxy- -xylulose (1-DX) and 4-hydroxy-
.
It was found by the tracer experiments that
+
D
L
-
which requires only NAD in PNP formation from
6
,7)
threonine (4-HT). The mechanism of PN formation
from 1-DX and 4-HT has been studied as the most in-
1-DXP and 4-PHT.
First, in vitro synthesis of vitamin B from 1-DX
6
teresting pathway in the biosynthesis of vitamin B
6
,
and 4-HT was examined in the presence of a range of
diŠerent coenzymes using a crude enzyme solution
prepared from cells in a buŠer without a stabilizer,
which is assumed to be advanced by a hypothetical
3
)
seven-step chemical sequence. Recently, researchers
Fig. 1. Minimum Reaction Scheme for the Synthesis of Pyridoxine (3) from 1-Deoxy-
Rhizobium
D-xylulose (1) and 4-Hydroxy-L-threonine (2) in
.
†
To whom correspondence should be addressed. Fax: +81-467-45-6812; E-mail: masaaki.tazoe
Abbreviations: PN, pyridoxine; 1-DX, 1-deoxy- -xylulose; 4-HT, 4-hydroxy- -threonine; PNP, pyridoxine 5
deoxy- -xylulose 5-phosphate; 4-PHT, 4-(phosphohydroxy)- -threonine
＠
roche.com
D
L

-phosphate; 1-DXP, 1-
D
L

In Vitro Synthesis of Vitamin B
6
935
however, synthesis of vitamin B
Therefore, we tried adding a stabilizer such as
glycerol or sucrose to the buŠer for cell disruption.
6
did not occur at all.
Table 1. EŠects of MnCl
2
and MgCl
System on Synthesis of Vitamin B
2
in the Complete Enzyme
6
from 1-Deoxy- -xylulose and
D
4
-Hydroxy-
L
-threonine
The complete enzyme system contained 80 m
M
Tris-HCl, pH
R. meliloti IFO 14782 was cultured at 28
production medium described in a previous paper,
then 3-d cells were harvested and washed twice with
.85 (w v) NaCl solution. The resulting cells were
suspended in 10 m
9
C in a
+
7
.5, 2.5 m
M
1-DX, 2.5 m
ATP, and crude enzyme solution (1.1 mg protein).
C for 2 h in a glass
M
4-HT, 0.38 m
M
NADP , 0.38 m
M
8
)
+
NAD , 5 m
M
Incubation was done without shaking at 28
tube (13 100 mm).
9
×
0
z
W
M
Tris-HCl, pH 7.5, with glycerol
Reaction mixture
Complete enzyme system
6
Vitamin B (nmol)
[
(
0, 5, 10, or 20
z
600
(w v)] or sucrose [3.75, 7.5, or 15z
W
0.46
0.56
0.58
2.77
w v)] (ˆnal A 250) and passed through a French
＝
W
plus 8.4 m
plus 32 m
plus 8.4 m
M
MnCl
MgCl
MnCl
2
pressure cell. The cell debris was removed by cen-
trifugation, and then the supernatant was fractionat-
M
2
M
2
and 32 m
M MgCl
2
ed with 80
precipitate was resuspended in 10 m
.5, desalted with the same buŠer, and used as an en-
z
(w v) ammonium sulfate. The
W
M
Tris-HCl, pH
7
zyme source of the following reaction mixture. The
reaction mixture contained 1-DX and 4-HT as sub-
strates, NAD , NADP , and ATP as coenzymes,
and the crude enzyme solution in Tris-HCl buŠer,
pH 7.5. After incubation of the reaction mixture at
a complete enzyme system). As shown in Table 1, ad-
dition of 8.4 m MnCl or 32 m MgCl stimulated
vitamin B formation. The concentration of vitamin
formation was increased by the addition of both
8.4 m MnCl and 32 m MgCl to a six-fold higher
yield than that in the complete enzyme system
without the supplement. The vitamin B synthesized
was analyzed by thin-layer chromatography (Silica
gel 60, Merck, chloroform methanol 3 1,v v) and
M
2
M
2
+
+
6
B
6
M
2
M
2
2
8
9
C for 2 h, the reaction was stopped by heating the
mixture for 3 min in a boiling water bath. The vita-
min B synthesized was measured by the turbidity
method with S. carlsbergensis ATCC 9080 as de-
6
6
＝
W
W W
8
)
scribed in a previous paper. From the study, appar-
ent synthesis of vitamin B was observed only when
the reaction mixture contained crude enzyme solu-
tion prepared from cells suspended in 10 m Tris-
HCl, pH 7.5, with 15 sucrose as an enzyme source.
Furthermore, in experiments with concentrations of
sucrose (zero to 17.5 ) in 10 m Tris-HCl, pH 7.5,
for cell disruption, the maximum vitamin B synthe-
sis was observed at the concentration of 15
sucrose, and could not be replaced by addition of
–20 glycerol to the buŠer. Adding a high concen-
tration of sucrose, 15 , to the buŠer for cell disrup-
tion was eŠective for the synthesis of vitamin B from
-DX and 4-HT, and might be for stabilization of en-
identiˆed as PN by bioautograms on an agar plate
with S. carlsbergensis ATCC 9080 (data not shown).
In E. coli, PNP is synthesized from 1-DXP and
4-PHT in the presence of only one coenzyme,
6
M
+
z
NAD , by PdxA and PdxJ proteins, as reported by
5
–7)
Cane and Laber et al
.
However, in Rhizobium
,
+
+
z
M
three coenzymes, NAD , NADP , and ATP, are es-
sential for in vitro synthesis of PN from 1-DX and
4-HT, although substrates and the product were
dephosphorylated forms of 1-DXP, 4-PHT, and
PNP, respectively. Establishment of the in vitro sys-
tem in R. meliloti will be valuable not only for isola-
tion of enzymes involved in formation of PN from
1-DX and 4-HT but also for deˆnition of the PN syn-
thetic pathway from 1-DX and 4-HT in Rhizobium.
6
z
5
z
z
6
1
zymes involved in the synthesis, but such a stabilizer
was not added to the buŠer for cell disruption in iso-
7
)
lation of the E. coli PdxA and PdxJ proteins.
References
+
+
The requirement of NAD , NADP , and ATP
and the optimum concentrations were examined
using a crude enzyme solution prepared from cells
1
2
)
)
Lam, H.-M. and Winkler, M. E., Metabolic relation-
ships between pyridoxine (vitamin B ) and serine
6
biosynthesis in Escherichia coli K-12. J. Bacteriol.
,
suspended in 10 m
M
Tris-HCl, pH 7.5, with 15+
z
172, 6518–6528 (1990).
sucrose. From the study, three coenzymes, NAD ,
Kennedy, I. A., Hill, R. E., Pauloski, R. M., Sayer, B.
G., and Spenser, I. D., Biosynthesis of vitamin B : ori-
gin of pyridoxine by the union of two acyclic precur-
sors, 1-deoxy- -xylulose and 4-hydroxy- -threonine.
J. Am. Chem. Soc., 117, 1661–1662 (1995).
+
NADP , and ATP were essential for in vitro synthe-
6
sis of vitamin B
6
from 1-DX and 4-HT, and their op-
+
timum concentrations were 0.38 m
M
for NAD and
D
L
+
NADP and 5 m
M
for ATP. The result suggested
that at least three enzymes might be involved in
3) Spenser, I. D. and Hill, R. E., The biosynthesis of
pyridoxine. Natural Product Reports, 12, 555–565
(1995).
formation of vitamin B
meliloti.
6
from 1-DX and 4-HT in R.
4
)
Hill, R. E., Himmeldirk, K., Kennedy, I. A.,
Pauloski, R. M., Sayer, B. G., Wolf, E., and Spenser,
EŠects of MnCl
2
and MgCl
2
on vitamin B forma-
6
tion were tested in 500
ing 80 m Tris-HCl, pH 7.5, 2.5 m
-HT, 0.38 m NADP , 5 m
m
l of reaction mixture contain-
6
I. D., The biogenetic anatomy of vitamin B . J. Biol.
M
M
1-DX, 2.5 m
M
+
+
Chem., 271, 30426–30435 (1996).
Cane, D. E., Hsiung, Y., Cornish, J. A., Robinson, J.
4
M
NAD , 0.38 m
M
M
5
)
ATP, and crude enzyme solution (1.1 mg protein, as

9
36
M. TAZOE et al.
K., and Spenser, I. D., Biosynthesis of vitamin B
6
: the
Spenser, I. D., Biosynthesis of vitamin B
6
: enzymatic
oxidation of 4-(phosphohydroxy)-
L
-threonine by
conversion of 1-deoxy- -xylulose-5-phosphate to
D
PdxA. J. Am. Chem. Soc., 120, 1936–1937 (1998).
Laber, B., Maurer, W., Scharf, S., Stepusin, K., and
pyridoxol phosphate. J. Am. Chem. Soc., 121,
6
7
)
)
7722–7723 (1999).
Schmidt, F. S., Vitamin B
pyridoxine 5
threonine and 1-deoxy-
PdxA and PdxJ protein. FEBS Lett., 449, 45–48
1999).
Cane, D. E., Du, S., Robinson, J. K., Hsiung, Y., and
6
biosynthesis: formation of
-phosphate from 4-(phosphohydroxy)-
-xylulose-5-phosphate by
8) Tazoe, M., Ichikawa, K., and Hoshino, T., Produc-
?
L
-
6
tion of vitamin B in Rhizobium. Biosci. Biotechnol.
D
Biochem., 63, 1378–1382 (1999).
9) Tazoe, M., Ichikawa, K., and Hoshino, T., Biosynthe-
(
sis of vitamin B
6
in Rhizobium. J. Biol. Chem., 275,
11300–11305 (2000).