ORIGINAL PAPER

Pusillimonas sp. 5HP degrading 5-hydroxypicolinic acid

Laimonas Karvelis • Renata Gasparaviciut _e •

Algirdas Klimavicius • Regina Jancien _e •

Jonita Stankeviciut _e • Rolandas Meskys

Received: 27 September 2012 / Accepted: 25 March 2013

� Springer Science+Business Media Dordrecht 2013

Abstract A bacterial strain 5HP capable of degrading

and utilizing 5-hydroxypicolinic acid as the sole source

of carbon and energy was isolated from soil. In addition,

the isolate 5HP could also utilize 3-hydroxypyridine

and 3-cyanopyridine as well as nicotinic, benzoic and

p-hydroxybenzoic acids for growth in the basic salt media.

On the basis of 16S rRNA gene sequence analysis, the

isolate 5HP was shown to belong to the genus Pusilli-

monas. Both the bioconversion analysis using resting cells

and the enzymatic assay showed that the degradation of

5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic

acid was inducible and proceeded via formation of the

same metabolite, 2,5-dihydroxypyridine. The activity of a

novel enzyme, 5-hydroxypicolinate 2-monooxygenase,

was detected in the cell-free extracts prepared from

5-hydroxypicolinate-grown cells. The enzyme was par-

tially purified and was shown to catalyze the oxidative

decarboxylation of 5-hydroxypicolinate to 2,5-dihydr-

oxypyridine.Theactivity of 5-hydroxypicolinate2-mono-

oxygenase was dependent on O2, NADH and FAD.

Keywords 5-Hydroxypicolinic acid �3-Hydroxypyridine � Degradation �3-Hydroxypyridine � 5-Hydroxypicolinate

2-monooxygenase � Pusillimonas

Introduction

The pyridine ring is a major constituent of natural

compounds including plant alkaloids, coenzymes and

antibiotics. Pyridine and its derivatives are used as

dyes, industrial solvents, herbicides, and pesticides.

The biodegradation of pyridine-containing com-

pounds has been studied intensively (Houghton and

Cain 1972; Kaiser et al. 1996; Fetzner 1998; Brandsch

2006; Li et al. 2010).

Catabolism of pyridine, especially the initial steps

of hydroxylation of monocarboxylated pyridines such

as 2-carboxypyridine (picolinic acid) (Tate and

Ensign 1974; Siegmund et al. 1990; Kiener et al.

1993), 3-carboxypyridine (nicotinic acid) (Nagel and

Andreesen 1989; Jimenez et al. 2008), and 4-carb-

oxypyridine (isonicotinic acid) (Singh and Shukla

1986) have been studied in detail. Nicotinate dehy-

drogenases, enzymes that catalyze the hydroxylation

of nicotinate, were purified from Bacillus niacini

(Nagel and Andreesen 1990), Pseudomonas fluores-

cens TN5 (Hurh et al. 1994), Eubacterium barkeri

(Gladyshev et al. 1996) and Ralstonia/Burkholderia

strain DSM 6920 (Schrader et al. 2002). In addition,

such enzymes as isonicotinate dehydrogenase and

L. Karvelis � R. Gasparaviciut _e � J. Stankeviciut _e �R. Meskys (&)

Department of Molecular Microbiology and

Biotechnology, Institute of Biochemistry, Vilnius

University, Mokslininku 12, LT-08662 Vilnius, Lithuania

e-mail: rolandas.meskys@bchi.vu.lt

A. Klimavicius � R. Jancien _eDepartment of Bioorganic Compounds Chemistry,

Institute of Biochemistry, Vilnius University,

Mokslininku 12, LT-08662 Vilnius, Lithuania

123

Biodegradation

DOI 10.1007/s10532-013-9636-3

picolinic acid 6-hydroxylase have been purified from

Mycobacterium sp. INA1 (Kretzer et al. 1993) and

Arthrobacter picolinophilus (Tate and Ensign 1974)

respectively. Moreover, it was shown that in Alcalig-

enes sp. strain UK21 6-hydroxypicolinic acid may be

also formed via conversion of quinolinic acid (Uchida

et al. 2003).

The microbial conversion of hydroxylated carb-

oxypyridines, particularly catabolism of hydroxypi-

colinic acids, has been less studied. The first step in the

degradation of nicotinic acid by both aerobic and

anaerobic organisms appears to be the same, i.e., the

formation of 6-hydroxynicotinic acid, but thereafter

the pathways diverge (Kaiser et al. 1996). The

anaerobic bacteria degrade 6-hydroxynicotinate by

reducing pyridine ring via formation of 1,4,5,6-

tetrahydro-6-oxonicotinate (Alhapel et al. 2006).

Bacillus and Pseudomonas spp. degrade 6-hydroxy-

nicotinate by an oxidative decarboxylation producing

2,5-dihydroxypyridine (Nagel and Andreesen 1990;

Nakano et al. 1999; Jimenez et al. 2008). The ring of

2,5-dihydroxypyridine is then cleaved oxidatively to

yield maleamic and formic acids (Jimenez et al. 2008).

The cells of the Bacillus sp. oxidize nicotinic acid via

6-hydroxynicotinic acid, 2,6-dihydroxynicotinic acid

and 2,3,6-trihydroxypyridine followed by ring cleav-

age and the formation of maleamic acid (Ensign and

Rittenberg 1964; Hirschberg and Ensign 1971). Such

compounds as 6-hydroxypicolinic, 3,6-dihydroxypi-

colinic acids and 2,5-dihydroxypyridine have been

detected as the main metabolites during picolinic acid

biodegradation by Bacillus sp. (Shukla 1975). How-

ever, the enzymes involved in conversion of 6-hy-

droxypicolinic acid have not been characterized so far.

The 5-hydroxypicolinic acid is a natural product

produced by Nocardia spp. (Makar’eva et al. 1989).

Some antibiotics, such as rubradirin and nikkomycin,

contain the fragment of 5-hydroxypicolinic acid (Kim

et al. 2008; Moon and van Lanen 2010). The hydroxy

derivatives of picolinic acid can be formed by

degradation of aminophenols (He and Spain 2000).

However, no data concerning microorganisms capable

to utilize 5-hydroxypicolinic acid or enzymes involved

in catabolism of this compound has been pub-

lished yet.

Oxidoreductases exhibiting the chemo- and regio-

selective hydroxylation of the pyridine ring represent a

perspective new and mild synthetic route to substi-

tuted pyridinols, many of which are potential drugs or

agrochemicals. (Yoshida and Nagasawa 2000; Wang

et al. 2005; Garrett et al. 2006). In this study, the

isolation and characterization of a new 5-hydroxypi-

colinic acid-degrading bacterium belonging to the

genus Pusillimonas is presented. A novel enzyme,

5-hydroxypicolinate 2-monooxygenase, catalyzing

the oxidative decarboxylation of 5-hydroxypicolinate

to 2,5-dihydroxypyridine is described.

Materials and methods

Chemicals

Picolinic, 3-hydroxypicolinic, 6-hydroxypicolinic,

nicotinic, 6-hydroxynicotinic acids, 5-hydroxy-2-

methylpyridine, 3-hydroxypyridine were obtained

from Merck. Phenazine methosulphate (PMS), 2,6-

dichloroindophenol (DCPIP), nitro blue tetrazole

(NBT) and cytochrome c were from Sigma-Aldrich.

5-Hydroxypicolinic acid was from Apollo Scientific.

5-Hydroxy-2-methylpyridine was synthesized accord-

ing to Iqbal et al. (2009). 2,5-Dihydroxypyridine was

synthesized according to Behrman and Pitt (1958).

Media and growth conditions

For the bacteria isolation, mineral salt medium (MSM)

was used: (grams per liter) 8.8 KH2PO4, 1.0

(NH4)2SO4, the final pH was adjusted to 7.2. After

autoclaving (121 �C, 30 min) and cooling, the med-

ium was supplemented with (grams per liter) 0.15

MgSO4�7H2O and salt solution (10 ml per liter). Salt

solution (g/l): CaCl2�2H2O 2.0, MnSO4�4H2O 1.0,

FeSO4�7H2O 0.5, Na2MoO4�2H2O 0.5, all compo-

nents were dissolved in 0.1 N HCl.

For enzyme assay and biodegradation studies the

bacteria were grown in MSMYP medium (MSM

supplemented with (grams per liter) 0.9 yeast extract,

1.0 peptone, 0.6 beef extract) containing 0.15 % of the

appropriate substrate. After cultivation biomass was

collected by centrifugation (6,0009g, 10 min),

washed with 0.9 % NaCl and frozen (-20 �C) until

further use.

Screening of bacterial strains

Samples of soils were cultivated aerobically in 125 ml

flask containing 30 ml of MSM supplemented with

Biodegradation

123

5-hydroxypicolinic acid (1.5 g/l). Samples were incu-

bated in orbital shaker (180 rpm) at 30 �C for at least

4 days. Then 1 ml of the dispersed soil suspension was

transferred to flasks containing the fresh medium

supplemented with the same substrate and incubated

for an additional 4 days under the same conditions.

After cultivation, the aliquots were diluted and spread

on the MSM agar plates containing 0.15 % of

5-hydroxypicolinic acid. Colonies were selected and

purified by streaking repeatedly on the Nutrient agar

medium and the MSM agar plates containing 0.15 %

of 5-hydroxypicolinic acid.

Identification of a 5-hydroxypicolinic acid

degrading strain

DNA was extracted according to Woo et al. (1992).

The gene for 16S rRNA was amplified using universal

primers w001 and w002 (Godon et al. 1997). The PCR

product was purified using a DNA purification kit and

cloned into pTZ57R/T plasmid (Thermo Fisher Sci-

entific, Lithuania). The cloned DNA fragment was

sequenced at the Sequencing Centre of Institute of

Biotechnology, Vilnius University Lithuania.

Bioconversion using whole cells

The strain 5HP was grown in MSM medium supple-

mented with 0.15 % of an appropriate substrate

aerobically at 30 �C for 4 days. Cells were harvested

(6,0009g, 10 min), washed twice in 0.9 % NaCl.

Bioconversion was performed in 50 mM potassium

phosphate buffer, pH 7.2, containing 0.15 mM of

substrate. The cells were suspended in this solution and

the suspension was incubated aerobically at 25 �C. The

buffer solution containing the appropriate compound

without cells and a cell suspension without the

compound served as controls. Following the centrifu-

gation to remove the cells, the bioconversion was

estimated by recording absorbance at 220–360 nm at

regular time intervals.

Enzyme assays

The cell-free extracts were prepared by suspending cell

paste in 50 mM potassium phosphate buffer, pH 7.2. The

cells were broken by ultrasonic treatment 30 s at 22 kHz.

The cell debris was removed by centrifugation (13,0009g,

5 min). 2,5-Dihydroxypyridine 5,6-dioxygenase activity

was measured at 30 �C spectrophotometrically at 320 nm

(e320 = 5,200 cm-1 M-1) as described previously

(Jimenez et al. 2008) with modifications. The reaction

mixture contained: 50 mM potassium phosphate buffer

(pH 7.2), 0.05 mM 2,5-dihydroxypyridine, 0.005 mM

FeSO4, and an appropriate amount of protein or cell-free

extract in a total volume of 1 ml. One unit of activity was

defined as the amount of enzyme that catalyzed the

disappearance of 1 lmol of 2,5-dihydroxypyridine in

1 min. Activities of nicotinate dehydrogenase and

6-hydroxynicotinic acid 3-hydroxylase were analyzed as

described previously (Jimenez et al. 2008). One unit of

activity was defined as the amount of enzyme necessary to

oxidize 1 lmol of substrate per min. Activity of 5-hy-

droxypicolinate 2-monooxygenase was analyzed spectro-

photometrically at 340 nm (e340 = 6,200 cm-1 M-1) in

50 mM potassium phosphate buffer, pH 7.2 containing

1 mM FAD and 0.15 mM NADH at 30 �C. One unit of

activity was defined as the amount of enzyme necessary to

oxidize 1 lmol of NADH per min.

Purification of 5-hydroxypicolinate

2-monooxygenase

The cells were harvested by centrifugation and

suspended in 20 mM potassium phosphate buffer pH

7.0, and the cell extracts were prepared by ultrasonic

treatment as described previously. The cell extracts

from a 100-ml culture were purified using ToyoSreen

AF-Blue-650 M 5 ml column. The column was

equilibrated with 20 mM potassium phosphate buffer,

pH 7.0, and the proteins were adsorbed with the same

buffer. Elution was performed in two steps. Initially,

the column was washed with 10 ml of binding buffer

containing 20 % of sucrose. Then proteins were eluted

with 5 ml of binding buffer. The fractions containing

the active enzyme were pooled, concentrated by

ultrafiltration and applied to a 5 9 200 mm column

containing Source 15PHE resin (GE Healthcare). The

enzyme was eluted with a linear gradient of 0–0.5 M

of NaCl in 20 mM potassium phosphate buffer pH 7.2.

The fractions containing the active enzyme were

pooled and concentrated by ultrafiltration.

Zymography analysis

The samples were resolved by gel electrophoresis

at room temperature on a 10 % non-denaturing

polyacrylamide gel. After electrophoresis, the gels

Biodegradation

123

were washed with 20 mM potassium phosphate buffer

pH 7.2. Then 20 mM potassium phosphate buffer pH

7.2 containing 0.2 mM 5-hydroxypicolinic acid,

0.01 mM NADH, 0.005 mM PMS and 0.3 mM NBT

was poured onto the gel. The results were recorded

after incubation at 25 �C for 3 h. The reaction mixture

without 5-hydroxypicolinic acid served as a control.

De novo sequencing of proteins

Protein bands were visualized by staining the gel with

Coommassie Blue R250 and excised from the gel

using a razor blade. Each sample was purified as

described previously (Hellman et al. 1995). Tryptic-

digest from each gel slice was analyzed by 4000

QTRAP (AB SCIEX) mass spectrometry in linear ion

trap mode using information dependent acquisition

(IDA) and dynamic exclusion protocol. The acquisi-

tion method consisted of an IDA scan cycle including

the enhanced mass scan (EMS) as the survey scan,

enhanced resolution scan (ER) to confirm charge state

and six dependent enhanced product ion (EPI) scans

(MS/MS). With the threshold of the ion intensity at

100,000 counts per second (cps), the IDA criteria were

set to allow the most abundant ions in the EMS scan to

trigger EPI scans. Survey MS scan was set to mass

range from 400 to 1,400 m/z. Dynamic ion exclusion

was set to exclude precursor ions after their two

occurrences during 60s interval. Peak lists were

generated using Analyst software 1.4.2 (AB/Sciex,

USA).

LC–MS analysis

To the samples of medium or enzymatic reaction the

same volume of acetonitrile was added. The insoluble

particles were removed by centrifugation (15,0009g,

15 min). Liquid chromatography-mass spectrometry

(LC–MS) analyses were performed using HPLC

system (CBM-20A controller, two LC-2020AD

pumps, SIL-30AC auto sampler and CTO-20AC

column oven; Shimadzu, Japan) equipped with pho-

todiode array (PDA) detector (SPD-M20A Promi-

nence diode array detector; Shimadzu, Japan) and

mass spectrometer (LCMS-2020, Shimadzu, Japan)

equipped with an ESI source. The chromatographic

separation was conducted using a Hydrosphere C18

column, 4 9 150 mm2 (YMC, Japan) at 40 �C and a

mobile phase that consisted of 0.1 % formic acid

(solvent A) and methanol (solvent B) delivered in

gradient elution mode at a flow rate of 0.6 mL min-1.

The elution programme used was as follows: isocratic

0 % B for 0.5 min, from 0 to 60 % B over 4.5 min,

isocratic 60 % B for 0.1 min, from 60 to 0 % B over

0.1 min, isocratic 0 % B for 5 min. Mass scans were

measured from m/z 10 up to 500, at 350 �C interface

temperature, 250 �C DL temperature, ±4,500 V

interface voltage, neutral DL/Qarray, using N2 as

nebulizing and drying gas. Mass spectrometry data

was acquired in both the positive and in the negative

ionization mode. The data was analyzed using Lab-

Solutions LCMS software.

Nucleotide sequence accession number

and bioinformatical analysis

Phylogenetic and molecular evolutionary analyses

were conducted using MEGA version 5.05 (Tamura

et al. 2011). The DNA sequence has been deposited in

GenBank and can be accessed via the accession

number KC602498.

Results

Isolation and identification of bacterium capable

of utilizing 5-hydroxypicolinic acid

Several bacterial isolates from different soil samples

were screened for their ability to grow on a mineral

medium supplemented with 5-hydroxypicolinic acid.

The isolate 5HP producing a dark pigment when

grown on solid medium containing 5-hydroxypicoli-

nic acid was selected for further studies. The analysis

of 16S rRNA gene sequence revealed that the strain

5HP was related to Pusillimonas species and, together

with Pusillimonas noertemannii BN9 and Pusillimon-

as sp. N11, formed a distinct branch on a phylogenetic

tree (Fig. 1).

To determine the substrate range capability of

Pusillimonas sp. 5HP, a number of aromatic com-

pounds were tested as potential growth substrates. The

growth was analyzed both on solid and in liquid

media. The growth test revealed that the strain utilized

5-hydroxypicolinic acid as the single carbon and

energy source (Fig. 2). LC–MS analysis confirmed the

depletion of 5-hydroxypicolinic acid, and \15 % of

the initial substrate concentration was detected in the

Biodegradation

123

medium after 86 h of cultivation. Nicotinic, 6-hydroxy-

nicotinic, benzoic and p-hydroxybenzoic acids as

well as 3-hydroxypyridine, 2,5-dihydroxypyridine and

3-cyanopyridine also served as growth substrates for

Pusillimonas sp. 5HP. The other tested pyridine deriv-

atives, such as 5-hydroxy-2-methylpyridine, 5-hydroxy-

2-hydroxymethylpyridine, 5-hydroxy-2-cyanopyridine

or picolinic, 3-hydroxypicolinic, 6-hydroxypicolinic,

2-hydroxynicotinic, and isocinchomeronic acids, did not

support the growth of Pusillimonas sp. 5HP. The growth

of Pusillimonas sp. 5HP on 5-hydroxypicolinate and

3-hydroxypyridine containing media was accompanied by

an accumulation of a green pigment, which gradually

turned dark brown suggesting the formation of 2,5-

dihydroxypyridine and its subsequent spontaneous auto-

oxidation (Khanna and Shukla 1977; Karvelis and Meskys

2004).

Bioconversions using the whole cells

The conversion of 5-hydroxypicolinic acid, 3-hydroxy-

pyridine, nicotinic acid and 2,5-dihydroxypyridine by

the resting cells of Pusillimonas sp. 5HP pre-grown in

the presence of the appropriate compound was observed.

In contrast, no bioconversion was detected in non-

induced control cells under the same conditions and

time. The 5-hydroxypicolinic acid-induced cells pre-

cultivated for 40–86 h catabolized 5-hydroxypicolinic

acid (18.2 mmol h-1 mg-1 of biomass) and 2,5-dihydr-

oxypyridine (12.7 mmol h-1 mg-1 of biomass) but did

not convert nicotinic acid. The 3-cyanopyridine-induced

cells could metabolize 3-cyanopyridine, nicotinamide,

nicotinic and 6-hydroxynicotinic acids and 2,5-dihydr-

oxypyridine without a lag period by the rate of 1.85,

1.68, 1.06, 1.04 and 0.9 mmol h-1 mg-1 of biomass,

respectively. The nicotinic acid-induced cells con-

verted the same compounds, except 3-cyanopyridine.

Pusillimonas sp. 5HP pre-grown in the presence of

3-hydroxypyridine could utilize 3-hydroxypyridine and

2,5-dihydroxypyridine only by the rate of 0.6 and 0.9

mmol h-1 mg-1 of biomass, respectively. These results

showed that the induction of enzymes catalyzing

utilization of pyridine compounds requires the presence

of the appropriate substrate. Since 3-hydroxypyridine,

3-cyanopyridine, 5-hydroxypicolinate and nicotinate-

Fig. 1 Unrooted tree

showing the phylogenetic

relationships between 5HP

and related Pusillimonasspecies. The tree was

constructed using the

neighbour-joining method

based on a comparison of

*1,600 nucleotides. The

bar represents difference in

one nucleotide per 200 bp

Fig. 2 Growth of Pusillimonas sp. strain 5HP in MSMYP

medium with (closed circles) and without 5-hydroxypicolinic

acid (open circles). Squares concentration of 5-hydroxypicol-

inic acid in the cultivation medium

Biodegradation

123

grown cells consumed 2,5-dihydroxypyridine, it was

proposed that this compound is an intermediate metab-

olite generated during the degradation pathways of

pyridine derivatives listed above.

Enzyme assay

2,5-Dihydroxypyridine 5,6-dioxygenase, which is

crucial for catabolism of 2,5-dihydroxypyridine, had

been previously detected in various pyridine deriva-

tives-degrading bacteria (Gauthier and Rittenberg

1971a, b; Kaiser et al. 1996; Jimenez et al. 2008;

Tang et al. 2012). To test the ability of Pusillimonas

sp. 5HP to induce the production of the analogous

enzyme, the cell-free extracts obtained from 5-hy-

droxypicolinate, 3-hydroxypyridine, 3-cyanopyridine,

and nicotinate-grown-Pusillimonas sp. 5HP were

analyzed for their ability to oxidize 2,5-dihydroxy-

pyridine in the presence of Fe(II) ions. The test

revealed that 2,5-dihydroxypyridine was consumed by

all crude extracts, and the highest activity of 2,5-

dihydroxypyridine 5,6-dioxygenase was detected in

the nicotinic acid-induced cells (Table 1). The cells

grown in the presence of nicotinic acid and 3-cyano-

pyridine also showed the activity of nicotinate dehy-

drogenase, but only if potassium ferricyanide, PMS

(DCPIP), NBT or cytochrome c was used as an

electron acceptor. The activity of 6-hydroxynicotinic

acid 3-hydroxylase was also detected in these cell-free

extracts. However, no conversion of 3-hydroxypyri-

dine to 2,5-dihydroxypyridine was detected in the cells

of Pusillimonas sp. 5HP cultivated in the presence of

3-hydroxypyridine.

The 5-hydroxypicolinate-dependent oxidation of

NADH was observed in the cell-free extracts of the

cells grown in the medium containing 5-hydroxypi-

colinic acid. The zymogram of the cell-free extracts is

shown in Fig. 3. The activity of the enzyme increased

by *25 % after addition of FAD, but not FMN. No

activity was detected under anaerobic conditions, in

the presence of NADPH or in the cells cultivated

without 5-hydroxypicolinic acid. Since the synthesis

of 2,5-dihydroxypyridine 5,6-dioxygenase was

induced in the cells of Pusillimonas sp. 5HP during

growth on 5-hydroxypicolinic acid, it was proposed

that 2,5-dihydroxypyridine may be a conversion

product of 5-hydroxypicolinic acid. To confirm this

hypothesis, the purified 2,5-dihydroxypyridine 5,6-

dioxygenase from Sinorhizobium sp. L1 (Karvelis and

Meskys 2004) was added into the reaction mixture

after completion of 5-hydroxypicolinic acid-depen-

dent NADH oxidation. A decrease in absorbance at

320 nm, the maximum of absorbance of 2,5-dihydr-

oxypyridine, was observed. In addition, an LC–MS

analysis of the reaction products was carried out as

described in Materials and methods. A new compound

(retention time–5.26 min,absorbtion maxima (nm)—

225 and 320, mass of ([M?H]?—112.10) that was

detected after the enzymatic conversion of 5-hydrox-

ypicolinic acid exactly matched to the authentic

2,5-dihydroxypyridine. Hence, it was assumed that

5-hydroxypicolinate 2-monooxygenase (decarboxy-

lating) was the enzyme involved at the initial stage

of catabolism of 5-hydroxypicolinic acid in Pusilli-

monas sp. 5HP cells.

The 5-hydroxypicolinate 2-monooxygenase selec-

tivity to FAD and NADH suggested that the protein

could specifically bind to Cibacron Blue F3GA dye.

The enzyme was purified from cell-free extract using

the affinity Blue and anion exchange resin. The

partially purified enzyme (fivefold purification; spe-

cific activity 0.5 U/mg of protein) was obtained

(Fig. 4). The enzyme was unstable and further

attempts to improve the purification procedure were

unsuccessful. The protein, after PAGE fractionation

(Fig. 4), was subjected to de novo sequencing. One

peptide with the amino acid sequence VGFL(I)PEA-

L(I)VGR was identified. BLAST analysis revealed

that the peptide has sequence homology to various

salicylate 1-monooxygenase related proteins, and

shows the highest similarity to a putative salicylate

1-monooxygenase from Burkholderia xenovorans

LB400 (YP_555487).

Discussion

2,5-Dihydroxypyridine was identified as an interme-

diate in the microbial degradation of nicotinate more

than six decades ago (Behrman and Stanier 1957). At

least four different degradation pathways, including

catabolism of 2-hydroxypyridine, 3-hydroxypyridine,

picolinic acid and nicotinic acid depends on formation

of this intermediate (Kaiser et al. 1996). Here, a new

degradation pathway found in Pusillimonas sp. 5HP is

described. These bacteria are capable to utilize

5-hydroxypicolinate via 2,5-dihydroxypyridine for-

mation. In addition, Pusillimonas sp. 5HP can utilize

Biodegradation

123

3-hydroxypyridine and nicotinic acid as a single

carbon and energy source. In all three cases, the

activity of an inducible 2,5-dihydroxypyridine 5,6-

dioxygenase has been observed. Hence, the catabolism

of these compounds proceeds through formation of

2,5-dihydroxypyridine (Fig. 5). It has been reported

that Achromobacter and Sinorhizobium spp. can

transform 3-hydroxypyridine to 2,5-dihydroxypyri-

dine, however a corresponding enzymatic activity has

not been detected in the induced cells (Houghton and

Cain 1972; Karvelis and Meskys 2004). Repeated

attempts to register oxidation of 3-hydroxypyridine by

adding NADP(H), NAD(H), methylene Blue, DCPIP,

NBT, FAD, FMN or their combination into the cell-

free extracts or membrane fraction of Pusillimonas sp.

5HP have been unsuccessful. However, the activity of

NADH-dependent 5-hydroxypicolinate 2-monooxy-

genase has been detected in 5-hydroxypicolinate-

induced cells of Pusillimonas sp. 5HP. The analogous

oxidative decarboxylation is catalyzed by salicylate

1-monooxygenase (Yamamoto et al. 1965) and 6-hy-

droxynicotinate 3-monooxygenases (Nakano et al.

1999; Jimenez et al. 2008). De novo sequencing of the

partially purified 5-hydroxypicolinate 2-monooxygen-

ase allowed identification of a single peptide which

shows sequence similarity to various enzymes belong-

ing to the group of salicylate 1-monooxygenases.

Hence, we demonstrate that5-hydroxypicolinate 2-mono-

oxygenase is a novel decarboxylating monooxygenase.

Conclusion

This is the first report of the biological conversion of

5-hydroxypicolinic acid. A 5-hydroxypicolinic acid-

degrading bacterium Pusillimonas sp. 5HP has been

isolated from the soil. The strain 5HP displays a broad-

Table 1 The enzyme activities in the cell-free extracts of Pusillimonas sp. 5HP

Substrate Enzyme activity, mU/mg of protein

5-Hydroxypicolinate

2-monooxygenase

2,5-Dihydroxypyridine

5,6-dioxygenase

Nicotinate

dehydrogenase

6-Hydroxynicotinic

acid 3-hydroxylase

Succinate \1 \1 \1 \1

5-Hydroxypicolinic acid 93 ± 9 687 ± 23 \1 \1

3-Hydroxypyridine \1 527 ± 61 \1 \1

3-Cyanopyridine \1 1416 ± 87 1423 ± 108 37 ± 5

Nicotinic acid \1 1872 ± 78 2364 ± 144 45 ± 13

The cells were grown in the MSMYP medium supplemented with 0.15 % of the appropriate substrate for 84 h. The activity of

5-hydroxypicolinate 2-monooxygenase was measured in the presence of NADH without addition of FAD. The activity of nicotinate

dehydrogenase was analysed using potassium ferricyanide

Fig. 3 Non-denaturing

PAGE (10 %) of cell-free

extracts of Pusillimonas sp.

5HP grown in the presence

of 5-hydroxypicolinic acid

and zymography of a

5-hydroxypicolinic acid-

oxidizing enzyme. Lane 1staining mixture without

5-hydroxypicolinic acid

(control), lane 2 staining

mixture with

5-hydroxypicolinic acid,

lane 3 protein molecular

mass marker

Fig. 4 SDS PAGE analysis of partially purified 5-hydroxypi-

colinate 2-monooxygenase. Lane 1 protein molecular mass

markers, lane 2 10 lg of partially purified 5-hydroxypicolinate

2-monooxygenase. Asterisk marks the protein band applied for

de novo sequencing

Biodegradation

123

spectrum degradation ability, it not only has the

enzymes participating in catabolism of 5-hydroxypi-

colinic acid but it can also consume 3-hydroxypyri-

dine and 3-cyanopyridine as well as nicotinic, benzoic

and p-hydroxybenzoic acids as a single carbon and

energy source. A novel enzyme catalyzing the oxida-

tive decarboxylation of 5-hydroxypicolinate to 2,5-

dihydroxypyridine has been identified.

Acknowledgments This research was funded by a Grant (No.

MIP-076/2011) from the Research Council of Lithuania. Authors

thanks dr. M. Ger for assisting with de novo sequencing and dr.

L. Kaliniene for critical reading of the manuscript.

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