2
R. Rahmanpour et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e5
(
5' CACC GTG AGT GAA AGC GGC GGC ACA TTC 3') and Reverse
50 mm, 4.6 mm) on a Hewlett-Packard Series 1100 analyzer, at a
primers (5' TCA GTC GTG GAC GTG CAA CGG C 3') for the DLDH gene
were designed with the intention of cloning into a pET151/D-TOPO
vector. The gene was amplified from genomic DNA by polymerase
chain reaction using Pfx Taq polymerase, using 34 cycles of: 30 s at
flow rate of 0.5 mL/min, with monitoring at 270 nm. The gradient
2 2
was as follows: 50% MeOH/H O for 5 min; 50e80% MeOH/H O from
5 to 12 min; 80% MeOH/H O from 12 to 25 min; 80-30% MeOH/H O
2
2
from 25 to 30 min. Peaks of interest were collected for further
analysis by mass spectrometry and MS-MS fragmentation.
ꢀ ꢀ ꢀ
9
4 C, 30 s at 58 C; 90 s at 72 C. The PCR product (1.4 kb) was
excised from a 1% agarose gel and extracted using a GeneJET PCR
Purification Kit (Thermo Scientific), and cloned into a pET151/D-
TOPO vector (Invitrogen) using manufacturer's instructions, and
transformed into One Shot TOP10 competent cells (Invitrogen). The
DNA sequence was confirmed via DNA sequencing.
3. Results
T. fusca dihydrolipoamide dehydrogenase was cloned from
genomic DNA, and was expressed as a His fusion protein from a
6
pET151 vector in Escherichia coli. The recombinant protein
expressed well as a 48 kDa protein, was purified by metal affinity
chromatography, and the fusion tag removed by digestion with TEV
protease (see Supplemental Fig. S2). Addition of riboflavin to the
growth media was found to yield recombinant enzyme with stoi-
2
.2. Overexpression of recombinant T. fusca dihydrolipoamide
dehydrogenase
A 1L culture of T. fusca DHLDH pET151/TOP10 in Luria-Bertani
ꢀ
broth containing 50
mg/ml ampicillin was grown at 37 C for
chiometric amounts of flavin cofactor (lmax 460 nm, see
2
e3 h. At OD595 ¼ 0.6, riboflavin (0.5 mM) and IPTG (0.5 mM) were
Supplemental Fig. S3). The recombinant enzyme was catalytically
added to the culture, which was then incubated in a shaker over-
night at 15 C at 200 rpm. After centrifugation at 6000g, the cell
active for NADH-dependent reduction of lipoic acid, with kcat 0.14
ꢀ
ꢁ1
s
m
and K 430 mM, and showed maximal activity at pH 6.0. The
pellet was re-suspended in 10 ml of lysis buffer (50 mM NaH
pH 8.0, 300 mM NaCl, 10 mM imidazole). Cells were lysed using a
cell disruptor at 21 kpsi. The cell lysate was then centrifuged at
2
PO
4
enzyme also showed NADH-dependent quinone reductase activity
ꢁ1
with p-benzoquinone (kcat 0.42 s
m
K 540 mM, see Supplemental
Figs. S4eS6), comparable with activity reported for dihy-
drolipoamide dehydrogenase from Streptomyces seoulensis [22].
We have previously reported that Dyp-type peroxidase DypB
13,000 g for 40 min. DLDH was then purified using nickel affinity
2
þ
chromatography. The supernatant was incubated with 2 ml of Ni
ꢀ
2þ
Sepharose beads at 4 C for 60 min. This cell supernatant-Ni
Sepharose mixture was then placed in disposable plastic column
and the resin washed with 50 ml of wash buffer (50 mM NaH PO
pH 8.0, 300 mM NaCl, 20 mM imidazole), then DLDH was eluted
with 7 ml of elution buffer (50 mM NaH PO pH 8.0, 300 mM NaCl
-
from Rhodococcus jostii RHA1 is able to oxidatively cleave b-aryl
ether lignin model compound 1 [17]. Treatment of 1 with peroxi-
dase Dyp1B from Pseudomonas fluorescens [23] or Thermobifida
fusca Dyp peroxidase [24] leads to the formation of a new peak
observed by C18 reverse phase HPLC at retention time 15.5 min (see
Fig. 1, trace B). Analysis of this peak by electrospray mass spec-
trometry gave m/z 660.4, corresponding to an oxidative dimer of 1,
and further analysis of this species by MS-MS fragmentation has
identified this species as dimer 2 arising from oxidative dimeriza-
tion of 1 [24]. The formation of compound 2 therefore represented a
convenient model for oxidative repolymerisation of lignin
fragments.
2
4
2
4
and 250 mM imidazole). Fractions were collected and analyzed by
SDS-PAGE gel electrophoresis. Fractions containing DLDH (48 kDa
band) were pooled and incubated overnight at room temperature
with TEV protease (1 mg/ml) in order to remove the His tag. The
DLDH-TEV mixture was centrifuged at 13,000 g for 15 min, and the
supernatant was concentrated using a 10 kDa Millipore Centricon.
Native DLDH was purified by passing the solution through a 5 ml
HisTrap Ni2 resin FPLC column, then desalted using a PD10
þ
Addition of 210 nM T. fusca dihydrolipoamide dehydrogenase
desalting column.
(DHLDH) and 100
mM NADH to an incubation of 1.5 mM b-aryl ether
1
with P. fluorescens Dyp1B was found to completely prevent the
2
.3. Dihydrolipoamide dehydrogenase assay
formation of 2 (see Fig. 1 trace E, and Supplemental Fig. S7),
whereas addition of NADH alone had no effect (trace B). Treatment
of either the oxidised product 2 or lignin model compound 1 with
T. fusca DHLDH gave no change as observed by HPLC, indicating that
DHLDH acted upon an intermediate species formed during the
oxidation of 1 to 2. The disappearance of product 2 was found to be
dependent upon the concentration of NADH, showing partial
All assays were run in 50 mM sodium phosphate buffer pH 6.0 at
ꢀ
2
5
C, in the presence of 0.2 mM NADH. Enzyme kinetics were
measured through the decrease in the absorption spectrum of
NADH at 340 nm over 2 min, as previously described by Youn et al.
[22].
disappearance of 2 at 25 and 50
dimer 2 is formed in approximately 10% yield from 1 (trace A), and
prevention of dimerization occurs at 50e100 M NADH, the stoi-
mM NADH (traces C,D). Since the
2.4. HPLC assays
m
Catalytic action of DHLDH toward oxidised lignin model com-
chiometry of NADH required to prevent formation of 2 is in the
range 1e2 nmoles NADH/nmole of dimer 2.
pound was studied by reaction of model compound with Pseudo-
monas fluorescens Dyp1B enzyme in presence of T. fusca DLDH
enzyme and NADH. The reaction was performed in 50 mM phos-
The formation of 2 from 1 could be rationalised by dimerization
of a phenoxy radical formed from 1. In support of this mechanism,
the conversion of 1 to 2 was also observed upon addition of nitroxyl
radical reagent TEMPO to 1. The catalytic mechanism of dihy-
drolipoamide dehydrogenase is known to proceed via reduction of
an active site disulfide cysteine pair by reduced flavin [25,26]. We
therefore propose the catalytic cycle shown in Fig. 2, where the
reduced form of the active site disulfide reduces a phenoxy radical
intermediate via a 1-electron transfer, to generate a cysteine radical
intermediate. We have not observed any flavin semiquinone in-
termediate by pre-steady state kinetic analysis of T. fusca DHLDH,
therefore we propose a further 1-electron transfer to a phenoxy
radical, generating the oxidised active site disulfide, which is
ꢀ
phate buffer pH 6.0 at 25 C in presence of 0.4 mM model compound,
2
5e200
hydrogen peroxide. The following reactions were set up: 1) model
compound þ buffer þ H ; 2) model compound þ Dyp1B þ H
model compound Dyp1B NADH; 3) model
þ NADH þ DHLDH. The solutions were
L) were taken and
mM NADH, 560 nM Dyp1B, 210 nM DHLDH and 0.5 mM
O
2 2
2 2
O ;
þ
þ
H
2
O
2
þ
compound þ Dyp1B þ H
2 2
O
ꢀ
incubated at 25 C for 30 min. Aliquots (500
m
these fractions were analyzed by HPLC. Aliquots for HPLC were
mixed with CCl COOH (100%, w/v, 50 L), and the solution was then
centrifuged for 10 min at 10,000 rpm. HPLC analysis was conducted
using a Phenomenex Luna 5 m C18 reverse phase column (100 Å,
3
m
m
Please cite this article in press as: R. Rahmanpour, et al., Identification of an extracellular bacterial flavoenzyme that can prevent re-
polymerisation of lignin fragments, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.10.144
Rahmanpour, Rahman
