PLP catabolism: Identification of the 4-pyridoxic acid dehydrogenase gene in Mesorhizobium loti MAFF303099

Article abstract of DOI:10.1016/j.bioorg.2007.07.003

The function of the mlr6793 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 4-pyridoxic acid dehydrogenase, an enzyme involved in the catabolism of PLP (Vitamin B₆). This enzyme was overexpressed in Escherichia coli and characterized. 4-Pyridoxic acid dehydrogenase is a 33 kDa protein that catalyzes the four electron oxidation of 4-pyridoxic acid to 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, using nicotinamide adenine dinucleotide as a cofactor. The k_cat for NADH production is 0.01 s^-1. The K_M values for 4-pyridoxic acid and NAD are 5.8 and 6.6 μM, respectively.

Full text of DOI:10.1016/j.bioorg.2007.07.003

Available online at www.sciencedirect.com
Bioorganic Chemistry 35 (2007) 458–464
www.elsevier.com/locate/bioorg
Communications
PLP catabolism: Identification of the
-pyridoxic acid dehydrogenase gene in
Mesorhizobium loti MAFF303099
4
Tathagata Mukherjee, Cynthia Kinsland, Tadhg P. Begley *
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
Received 11 June 2007
Available online 14 September 2007
Abstract
The function of the mlr6793 gene from Mesorhizobium loti MAFF303099 has been identified.
This gene encodes 4-pyridoxic acid dehydrogenase, an enzyme involved in the catabolism of PLP
(
6
Vitamin B ). This enzyme was overexpressed in Escherichia coli and characterized. 4-Pyridoxic acid
dehydrogenase is a 33 kDa protein that catalyzes the four electron oxidation of 4-pyridoxic acid to 3-
hydroxy-2-methylpyridine-4,5-dicarboxylate, using nicotinamide adenine dinucleotide as a cofactor.
ꢀ1
The kcat for NADH production is 0.01 s . The K
.6 lM, respectively.
2007 Elsevier Inc. All rights reserved.
M
values for 4-pyridoxic acid and NAD are 5.8 and
6
ꢀ
Keywords: 4-Pyridoxic acid dehydrogenase; 4-Pyridoxic acid; 3-Hydroxy-2-methylpyridine-4,5-dicarboxylate;
Vitamin B
6
1
. Introduction
The bacterial pyridoxine (vitamin B ) catabolic pathway in Pseudomonas sp. MA-1 and
6
Mesorhizobium loti. is shown in Fig. 1 [1,2]. Of the 10 enzymes used for PLP catabolism,
only five of the corresponding genes have been identified. Recently, the genes encoding
pyridoxine-4-oxidase, 4-pyridoxolactonase, pyridoxal-4-dehydrogenase and 2-methyl-3-
*
Corresponding author. Fax: +1 607 255 4137.
E-mail address: tpb2@cornell.edu (T.P. Begley).
0
045-2068/$ - see front matter ꢀ 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.07.003

T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
459
OH
O
O
HO
OH
HO
N
N
Pyridoxine
1
.1.3.12
4
-pyridoxolactone
(I)
pyridoxal
-dehydro
(IV)
genase 1.1.1.107
pyridoxine
- oxidase
4-pyridoxo
lactonase
4
4
3.1.1.27
O
O
OH
O
HO
HO
O
HO
HO
O
HO
HO
O
HO
P
O
O
OH 3.1.3.74
HO
OH
1.2.3.8
OH
O
OH
N
pyridoxal
N
pyridoxal
oxidase
Pyridoxal 5-
phosphate
phosphatase
N
N
N
Pyridoxal
(III)
5-Formyl-3-hydroxy
3-Hydroxy-2-methyl
pyridine-4,5-dicarbo
xylate
(VII)
4-Pyridoxic Acid
(II)
-2-methylpyridine-4
(
V)
-carboxylate
(
VI)
O
O
HO
O
OH
HO
2
4.1.1.51
1.14.12.4
N
H
3.5.1.29
O
OH
Succinic
semialdehyde
3
-hydroxy-2-methylp
yridine-4,5-dicarboxy
late 4-decarboxylase
N
O
3
-hydroxy-2-methyl
pyridinecarboxylate
dioxygenase
O
OH
2-(acetamidomet
hylene)succinate
hydrolase
3
-Hydroxy-2-
-(Acetamidomet
hylene)succinate
methylpyridine
(X)
-
5-carboxylate
(IX)
(
VIII)
Fig. 1. The vitamin B
6
catabolic pathway in Pseudomonas sp. MA-1 and Mesorhizobium loti MAFF303099 [3].
The reactions shown in the box are catalyzed by 4-pyridoxic acid dehydrogenase.
hydroxypyridine-5-carboxylic acid oxygenase were identified in Mesorhizobium loti
MAFF303099 [4–7]. These genes are not part of an operon, but they are all close to each
1
other on the M. loti chromosome suggesting that other PLP catabolic genes might also be
found in this region. In particular, mlr6793, annotated as 3-hydroxybutyryl-CoA dehydro-
genase (EC 1.1.1.157) [8], is likely to be one of the three missing dehydrogenases. The 309
amino acid gene product is a NAD binding protein with calculated molecular weight of
3
3 kDa [9]. In this communication, we report the cloning and overexpression of
mlr6793 and demonstrate that the purified gene product has a dual dehydrogenase activity
catalyzing the 4-electron oxidation of 4-pyridoxic acid (compound V) to 3-hydroxy-2-
methylpyridine-4,5-dicarboxylate (compound VII).
2
. Materials and methods
2
.1. Materials
A dehydrated form of Luria–Bertani (LB) broth was purchased from EMB Chemicals
(
Gibbstown, NJ). Ampicillin and isopropyl-b-D-thiogalactopyranoside (IPTG) were
obtained from Lab Scientific Inc. (Livingston, NJ). 4-Pyridoxic acid, NAD, NADH, lyso-
zyme from chicken egg white, agar, and sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis (SDS–PAGE) molecular weight markers were from Sigma (St. Louis, MO).
Triethylamine was from Fisher (Fairlawn, NJ). TFA and methanol (HPLC grade), sodium
chloride and imidazole were from Acros Organics (NJ). Sodium dihydrogen phosphate
1
Abbreviations used: NAD, nicotinamide adenine dinucleotide; NADH, reduced form of NAD; FAD, flavin
adenine dinucleotide; TEA, triethylamine; TFA, trifluoroacetic acid; IPTG, isopropyl-b-D-thiogalactopyranoside;
LB, Luria–Bertani; PLP, pyridoxal-5-phosphate.

460
T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
monohydrate was from Mallinckrodt Baker Inc. (Phillipsburg, NJ). Deuterium oxide
D O) was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). Micro-
(
2
con YM-10 membranes (10,000 MWCO) and an Amicon Ultra centrifugal filter device
10,000 MWCO) were obtained from Millipore (Billerica, MA). The Supelcosil LC-18-T
(
column for HPLC was from Supelco (Bellefonte, PA). Escherichia coli strain MachI
and the Gateway system were from Invitrogen (Carlsbad, CA). Nucleospin Purification
kit, Phusion DNA polymerase, E. coli BL21(DE3) and the Ni–NTA superflow resin were
obtained from Macherey-Nagel (Easton, PA), New England Biolabs (Ipswich, MA),
Novagen (San Diego, CA) and Qiagen (Valencia, CA), respectively.
2
.2. Molecular cloning
Standard methods were used for DNA manipulations [10,11]. Plasmid DNA was puri-
fied with the Qiagen Miniprep kit and DNA fragments were purified from agarose gel with
the Nucleospin Purification kit. E. coli strain MachI was used as a recipient for transfor-
mations during plasmid construction and for plasmid propagation. Phusion DNA
polymerase was used for PCR following the manufacturer’s recommendations. The
pENTR-TEV-D-TOPO and the Gateway system were used following the manufacturer’s
instructions with slight modifications.
2
.3. Cloning of M. loti mlr6793
The M. loti mlr6793 gene was amplified from genomic DNA by PCR with the following
0
0
0
primer pair: 5 -CAC CAT GAT CCG AAA TAT CGC CAT CAT C-3 and 5 -CTA CTC
0
CCG GCC TTC CAG GAT GCG TC-3 . The PCR product was purified and used in a
topoisomerase mediated reaction with pENTR-TEV-D-TOPO essentially following the
manufacturer’s instructions. Clones were screened by PCR and verified by sequencing.
A correct clone was used in an LR recombination reaction with the plasmid pDESTF1,
which is a Gateway adapted vector based on the pET-system. The plasmid pDESTF1
encodes an N-terminal 6· His tag and is under the control of the T7lac promoter. Clones
were screened by restriction digestion. A correct clone was named pMl5337.XF1.
2
.4. Overexpression and purification
The plasmid pMl5337.XF1 was used to transform E. coli BL21(DE3). A starter culture
was prepared by growing a single colony of transformed cells in 10 ml of LB media con-
taining 100 lg/ml of ampicillin at 37 ꢁC with overnight agitation. One liter LB medium
(
20 g/L), containing 100 lg/ml of ampicillin, was inoculated with this starter culture.
The cells were grown at 37 ꢁC with shaking at 250 rpm until the culture reached an
OD590 of 0.6 at which point they were induced by adding IPTG to a final concentration
of 0.8 mM, the temperature was lowered to 15 ꢁC and the cells were allowed to grow
for a further 12 h. The cells were then harvested by centrifugation at 10,000g for 8 min
at 4 ꢁC.
Cells from 1 L of culture were re-suspended in 20 ml of binding buffer (50 mM
NaH PO , 150 mM NaCl, 10 mM imidazole, pH 7.7) and approximately 2 mg of lysozyme
2
4
was added. The cells were then lysed by sonication (Misonix Sonicator 3000, pulse ‘on’
time 1.0 s, pulse ‘off’ time 1.0 s, output level 8.0, 30 cycles) five times on ice. The cell debris

T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
461
was removed by centrifugation at 17,000 rpm for 40 min at 4 ꢁC. The clarified supernatant
was loaded on to a 5 ml Ni–NTA-affinity column pre-equilibrated with binding buffer kept
at 4 ꢁC. The Ni–NTA-affinity column was then washed with 100 ml wash buffer (50 mM
NaH PO , 150 mM NaCl, 20 mM imidazole, pH 7.7). The protein was eluted from the
2
4
column with elution buffer (50 mM NaH PO , 150 mM NaCl, 200 mM imidazole, pH
2
4
7
1
.7) at 4 ꢁC. The fractions containing protein were pooled and concentrated using YM-
0 Amicon Ultracentrifugal filters at 5000g to a final volume of 500 ll. The concentrated
sample was desalted into 100 mM phosphate buffer at pH 8.0 using an Econo-Pac 10DG
disposable chromatography column. The yield of the purified protein was 6 mg/L.
2
.5. Enzymatic assay based on NADH production
The reaction mixture consisted of: enzyme (5 lM), NAD (1 mM) and 4-pyridoxic acid
(
50 lM) in 100 mM potassium phosphate buffer at pH 8.0. The enzymatic activity was
monitored by measuring the increase in absorbance at 340 nm due to the production of
NADH. A control reaction without NAD showed negligible background activity.
2
.6. HPLC analysis
HPLC analysis of the enzymatic reaction mixture was performed on a Hewlett-Packard
1
100 instrument using a Supelcosil LC-18-T (15 cm · 4.6 mm, 3.0 lm) column. Solution A
contained water with 0.1% TFA, and solution B contained methanol with 0.1% TFA. The
following linear gradient mixing solution A with solution B was used: 100% solution A for
0
–2 min, 100–80% solution A from 2 to 12 min, 80–0% solution A in 12–13 min, 0–100%
solution A in 13–14 min and 100% solution A in 14–17 min. Flow rate was 1 ml/min,
absorbance was measured at 254 nm (characteristic for NAD) and 320 nm (characteristic
of 4-pyridoxic acid, compound V). Under these conditions the following compounds were
readily separated (retention time in parenthesis): 4-pyridoxic acid (compound V, 9.8 min),
3
-hydroxy-2-methylpyridine-4,5-dicarboxylate (compound VII, 7.35 min), NAD
(
8.75 min).
2
.7. Reaction time course
A time course was determined with a reaction mixture (1 ml) containing 17 mM NAD,
1
.5 mM 4-pyridoxic acid (compound V) and 150 lM of pure enzyme. At various time
points, 100 ll of the reaction mixture was quenched by addition to 100 ll of 10% TFA.
This mixture was filtered through Microcon YM-10 and 100 ll of the filtrate was analyzed
by HPLC.
2
.8. Product purification
A reaction mixture (5.5 ml) containing 78 mg of NAD (20 mM), 6 mg of 4-pyridoxic
acid (6 mM) and freshly purified enzyme (final concentration of 50 lM) was incubated
overnight at room temperature. It was then filtered through a YM-10 Microcon centrifu-
gal filter at 14,000g for 30 min and the filtrate was analyzed by HPLC. The enzymatic
product (retention time 7.35 min) was collected over multiple injections. Methanol was

462
T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
removed by rotary evaporation, TFA and water were removed under high vacuum over-
night. The resulting white powder was characterized by NMR and ESI-MS analysis.
2
.9. Steady state kinetic analysis
The steady state kinetic parameters for 4-pyridoxic acid dehydrogenase were deter-
mined by monitoring the absorbance at 370 nm over time. To a reaction mixture
500 ll) containing 243 nM enzyme and 400 lM of NAD, varying concentration of
(
4
1
-pyridoxic acid (V) was added. The rate of formation of NADH was monitored over
5 min at 370 nm for each concentration of the substrate. The extinction coefficient of
NAD in 100 mM phosphate buffer at pH 8.0 at 370 nm was determined to be
ꢀ
1
ꢀ1
2
600 M cm . The KM for 4-pyridoxic acid (V) and kcat for the production of
NADH by the enzyme was determined by fitting the rate of product formation versus
substrate concentration using non-linear regression to the Michaelis–Menten equation
using Grafit 5.0.11 (Erithacus Software Limited, Surrey, UK). In order to determine
the KM for NAD a similar experiment was done in which the reaction mixture
(
500 ll) contained 243 nM enzyme and 100 lM of 4-pyridoxic acid (V) and the concen-
tration of NAD was varied. All solutions were made in 100 mM phosphate buffer at
pH 8.0.
3
. Results and discussion
3
.1. Reaction time course
The formation of 3-hydroxy-2-methylpyridine-4,5-dicarboxylate (compound VII) and
the disappearance of 4-pyridoxic acid (compound V) as a function of time is shown in
2
1
1
1
1
1
000
800
600
400
200
000
V
5 min
2
4
6
9
1
0 min
0 min
0 min
0 min
80 min
VII
8
6
4
2
00
00
00
00
0
6
7
8
9
10
11
12
Time / min
Fig. 2. HPLC trace showing the disappearance of the reactant (retention time of 9.8 min) and the appearance of
the product (retention time of 7.35 min).

T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
463
Fig. 2. The absence of any other peaks at 320 nm, demonstrated that 5-formyl-3-hydroxy-
-methylpyridine-4-carboxylate (compound VI), the two-electron oxidation product of 4-
2
pyridoxic acid (compound V), did not accumulate during the reaction. Under the acidic
conditions used to quench the enzymatic reactions NADH is degraded to a non-chromo-
phoric compound at 320 nm and is therefore not detected in the HPLC-chromatogram,
monitored at 320 nm.
3
.2. Product characterization
The white colored compound obtained as a result of the enzymatic reaction mixture
was identified as 3-hydroxy-2-methylpyridine-4,5-dicarboxylate (compound VII) by
1
NMR and MS analysis. H NMR (300 MHz, D O) d 2.69 (s, 3H, CH ) and 8.42 (s,
2
3
1
H, C –H). Negative ion mode ESI-MS (Esquire-LC_00146 instrument, Bruker) showed
6
a species with m/z = 196, which corresponded to the calculated mass of mono anionic 3-
hydroxy-2-methylpyridine-4,5-dicarboxylate (compound VII). Fragmentation analysis
resulted in the formation of a species with m/z = 152 (single decarboxylation, Mꢀ44),
which when isolated and fragmented further, showed the formation of the species with
m/z = 108 (double decarboxylation, Mꢀ88).
3
.3. Steady state kinetic analysis
The steady state kinetic parameters for the enzyme were determined by monitoring the
production of NADH in a UV–visible spectrophotometer. The k for the NADH pro-
cat
ꢀ
1
duction is 0.01 s . The KM for 4-pyridoxic acid (compound V) (under saturating concen-
tration of NAD) is 5.8 (± 0.9) lM. The KM for NAD (under saturating concentration of 4-
pyridoxic acid, compound V) is 6.6 (± 1.3) lM. The absorbance for NADH production
was monitored at 370 nm instead of 340 nm (kmax for NADH) since both 4-pyridoxic acid
(
compound V) and 3-hydroxy-2-methylpyridine-4,5-dicarboxylate (compound VII) have
some absorbance at 340 nm.
4
. Conclusion
We have demonstrated that the mlr6793 gene product catalyzes the 4-electron oxidation
of 4-pyridoxic acid (compound V) to 3-hydroxy-2-methylpyridine-4,5-dicarboxylate (com-
pound VII) using NAD as a cofactor. The intermediate 5-formyl-3-hydroxy-2-methylpyri-
dine-4-carboxylate (compound VI) did not accumulate in the enzymatic reaction mixture.
This implies that the enzyme catalyzes the oxidation of VI to VII, faster than the oxidation
of V to VI. Since we see no reduction of NAD in absence of the substrate and the reaction
product has been unambiguously identified, it is reasonable to assume that two moles of
NAD are consumed for each mole of substrate oxidized. Sequence analysis indicates only
one nucleotide binding site so we assume that both hydride transfer reactions are occur-
ring at the same active site. The mlr6793 encoded pyridoxic acid dehydrogenase is different
from the previously characterized enzyme from Pseudomonas sp. MA-1 which uses FAD
as a cofactor and only catalyzes the 2-electron oxidation of 4-pyridoxic acid (compound
V) to 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylate (compound VI). FAD is not
required by the mlr6793 encoded pyridoxic acid dehydrogenase that has been character-
ized in this paper.

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T. Mukherjee et al. / Bioorganic Chemistry 35 (2007) 458–464
Acknowledgments
We are grateful to Dr. Yasunobu Ohkawa, Director of Genebank, National Institute of
Agrobiological Sciences, Ibaraki, Japan for providing us with the M. loti MAFF303090
strain. This research was supported by a grant from the National Institutes of Health
(
GM069618).
References
[
1] E.E. Snell, B.E. Haskell, in: M. Florkin, E.H. Stotz (Eds.), Comprehensive Biochemistry, vol. 21, Elsevier,
North Holland, New York, 1971, pp. 47–67.
[
[
[
[
2] R.W. Burg, V.W. Rodwell, E.E. Snell, J. Biol. Chem. 235 (1960) 1164–1169.
3] T. Yagi, G.M. Kishore, E.E. Snell, J. Biol. Chem. 258 (1983) 9419–9425.
4] B. Yuan, Y. Yoshikane, N. Yokochi, K. Ohnishi, T. Yagi, FEMS Microbiol. Lett. 234 (2004) 225–230.
5] J. Funami, Y. Yoshikane, H. Kobayashi, N. Yokochi, B. Yuan, K. Iwasaki, K. Ohnishi, T. Yagi, Biochim.
Biophys. Acta 1753 (2005) 234–239.
[
[
6] N. Yokochi, S. Nishimura, Y. Yoshikane, K. Ohnishi, T. Yagi, Arch. Biochem. Biophys. 452 (2006) 1–8.
7] B. Yuan, N. Yokochi, Y. Yoshikane, K. Ohinishi, T. Yagi, J. Biosci. Bioeng. 102 (2006) 504–510.
[8] http://theseed.uchicago.edu/FIG/protein.cgi?prot=fig|266835.1.peg.5337user=&48hr_job=&new_framework=0.
[
9] http://www.genome.ad.jp/dbget-bin/www_bget?mlo+mlr6793.
[
[
10] F.M. Ausubel, R. Brent, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1987.
11] J. Sambrook, E.F. Fritsch, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Plainview, New York, 1989.