In this study, an Agrobacterium tumefaciens gene encoding a puta-tive alternaputa-tive NADH dehydrogenase AtuNDH-2 was isolated and expressed in Escherichia coli as a His6-tagged protein
Trang 1Characterization and interactions with bacterial and thylakoid
membranes
Laetitia Bernard*,‡, Carine Desplats‡, Florence Mus†, Ste´phan Cuine´, Laurent Cournac and
Gilles Peltier
CEA Cadarache, Direction des Sciences du Vivant, De´partement d’Ecophysiologie Ve´ge´tale et Microbiologie des Bacte´ries et Microalgues, UMR 6191 CNRS-CEA, Aix-Marseille II, Saint-Paul-lez-Durance, France
Electrons that enter the respiratory chain can originate
from three different types of NADH dehydrogenase
Complex I (NDH-1), a multisubunit transmembrane
enzyme coupling quinone reduction to proton
translo-cation, is present in bacteria, as well as in plant, fungal
and mammal mitochondria [1] Multisubunit sodium-pumping NADH : quinone oxidoreductases are found
in some bacterial respiratory chains [2,3] Mono-meric type II NADH dehydrogenases (NDH-2), which contain a flavinic cofactor and are incapable of H+
Keywords
flavoenzyme; type II NADH dehydrogenase;
plastoquinone reduction; quinone reduction;
respiration
Correspondence
G Peltier, CEA Cadarache, Direction des
Sciences du Vivant, De´partement
d’Ecophysiologie Vegetale et Microalgues,
UMR 6191 CNRS-CEA, Aix-Marseille II,
F-13108 Saint-Paul-lez-Durance, France
Fax: +33 442 25 62 65
Tel: +33 442 25 76 51
E-mail: gilles.peltier@cea.fr
Present address
*UMR Microbiologie et Ge´ochimie des sols,
INRA ⁄ Universite´ de Bourgogne, Dijon,
France
†Department of Plant Biology, The Carnegie
Institution of Washington, Stanford, CA, USA
Note
‡These authors contributed equally to this
study
(Received 4 April 2006, revised 16 May
2006, accepted 9 June 2006)
doi:10.1111/j.1742-4658.2006.05370.x
Type II NADH dehydrogenases (NDH-2) are monomeric enzymes that cat-alyse quinone reduction and allow electrons to enter the respiratory chain
in different organisms including higher plant mitochondria, bacteria and yeasts In this study, an Agrobacterium tumefaciens gene encoding a puta-tive alternaputa-tive NADH dehydrogenase (AtuNDH-2) was isolated and expressed in Escherichia coli as a (His)6-tagged protein The purified
46 kDa protein contains FAD as a prosthetic group and oxidizes both NADH and NADPH with similar Vmax values, but with a much higher affinity for NADH than for NADPH AtuNDH-2 complements the growth (on a minimal medium) of an E coli mutant strain deficient in both NDH-1 and NDH-2, and is shown to supply electrons to the respiratory chain when incubated with bacterial membranes prepared from this mutant By measuring photosystem II chlorophyll fluorescence on thylak-oid membranes prepared from the green alga Chlamydomonas reinhardtii,
we show that AtuNDH-2 is able to stimulate NADH-dependent reduction
of the plastoquinone pool We discuss the possibility of using heterologous expression of NDH-2 enzymes to improve nonphotochemical reduction of plastoquinones and H2production in C reinhardtii
Abbreviations
DPI, diphenyleneiodonium; IPTG, isopropyl thio-b- D -galactoside; NDH-1, NADH dehydrogenase or Complex I; NDH-2, type II NADH
dehydrogenases; PQ, plastoquinone; PS I, photosystem I; PS II, photosystem II; ROS, reactive oxygen species; SOD, superoxide dismutase;
UQ, ubiquinone.
Trang 2pumping activity, have been evidenced in plant
mito-chondria [4–6], some bacteria [7–9], yeasts [10–12] and
more recently in archae [13] and protozoans [14]
Progress in systematic genome sequencing over the
last decade has revealed numerous putative NDH-2
sequences in various types of organisms [15]
Prokaryotic NDH-2s appear to play an important
role in both aerobic and anaerobic metabolism, and
ful-fil a high diversity of functions, depending on the
organ-ism and the environmental conditions For example,
in the aerobic nitrogen-fixing bacteria Azotobacter
vinelandii, a NDH-2 protects nitrogenase from O2
inhi-bition, by drastically increasing the respiration rate [16]
In the facultative aerobe Bacillus subtilis [17] or in the
obligatory fermentative aerotolerant Zymomonas
mobi-lis[18], NDH-2 has been reported to replace NDH-1 in
a simplified respiratory chain A NDH-2 has been
shown to mediate electron transfer to the
membrane-bound methane monooxygenase of the methanotroph
Methylococcus capsulatus [19] In cyanobacteria, the
existence of several NDH-2s has been reported
Although one of these was able to complement an
Escherichia coli-mutant deficient in NDH-1 and
NDH-2, they were proposed, because of their low
activ-ity, to have a sensor rather than a bioenergetic function
[20] In other bacteria, although the physiological role of
NDH-2 is still unclear, the NDH-1⁄ NDH-2 ratio seems
to be regulated as a function of variations in the growth
conditions [21]
In chloroplasts of higher plants and algae, in
addi-tion to the photosynthetic electron transfer chain
oxid-izing water at photosystem II (PS II) and reducing
NADP+ at photosystem I (PS I), the existence of a
respiratory chain including both nonphotochemical
reduction and oxidation of the plastoquinone (PQ)
pool has been shown [22] In higher plant chloroplasts,
dark PQ reduction is mediated by a multisubunit
com-plex homologous to bacterial comcom-plex I [23,24] In the
green unicellular alga Chlamydomonas reinhardtii such
a complex is absent from chloroplasts [22] Based on
pharmacological studies, the involvement of a
plasti-dial NDH-2 enzyme has been proposed [25,26]
Whether bacterial or mitochondrial NDH-2s, which
normally reduce ubiquinones (UQs), are able to reduce
PQs and interact with the photosynthetic electron
transport chain is not established
In this study, we report on the isolation of an
Agro-bacterium tumefaciens gene coding for a putative
NDH-2 (AtuNDH-2) Following expression in E coli,
a His-tagged protein was purified by nickel-affinity
chromatography The purified AtuNDH-2
recombin-ant protein is shown to complement growth on a
min-imal medium of an E coli mutant strain deficient in
both NDH-1 and NDH-2, and to supply electrons to the respiratory chain when incubated with bacterial membranes prepared from this mutant AtuNDH-2
is also shown to reduce PQs of the photosynthetic electron transport chain when incubated with C rein-hardtiithylakoids
Results
Sequence analysis of AtuNDH-2 The A tumefaciens genome, which is 60% GC rich, con-tains a unique protein sequence sharing common fea-tures with already described NDH-2 genes (NCBI accession number AI2824) The putative A tumefaciens
relatively poor identity with E coli (NDH), Saccharo-myces cerevisiae (NDE1) and Solanum tuberosum (StNDB1) protein sequences, respectively, 28, 26 and 25% The AtuNDH-2 sequence was compared with 49 NDH-2 or putative NDH-2 sequences from prokaryo-tes, fungi and plants using phylogenetic analysis (Fig 1) NDH-2 can be classified in four different groups, three of which contain prokaryotic NDH-2s The ‘prokaryote A’ subgroup includes most of the known eubacterial NDH-2s, including the E coli [27],
B subtilis[17] and A vinelandii [16] enzymes The ‘pro-karyote C’ subgroup contains cyanobacterial NDH-2 as well as plant NDC [6] AtuNDH-2 belongs to the poorly described ‘prokaryote B’ group containing eubacterial and cyanobacterial sequences The last subgroup con-tains plant NDA, NDB as well as yeast and C rein-hardtii sequences AtuNDH-2 shares from 24 to 28% identity and 40 to 48% similarity with rotenone-insensit-ive NAD(P)H dehydrogenases of plant mitochondria Alignment of representative NDH-2 protein sequences from the four different families (Fig 2) revealed high conservation in two domains showing most of the cri-teria for dinucleotide binding, including a bab fold and
a GxGxxG motif [28] Based on a comparison with the lipoamide dehydrogenase sequence (an enzyme which shares significant similarity with NDH-2s and the struc-ture of which has been resolved), the first binding site can be attributed to FAD and the second to NAD(P)H [27] The C-terminal domain of the protein, which con-tains 20 hydrophobic residues (Fig 2), has been sug-gested to anchor the enzyme to the membrane [29]
Expression, purification and biochemical characterization of AtuNDH-2
In order to express a His-tagged AtuNDH-2 protein
in E coli, the AtuNDH-2 gene sequence was amplified
Trang 3from genomic DNA, cloned into pSD80 with a
C-ter-minus (His)6-tag sequence The resulting plasmid was
used to transform E coli strain DH10b The
recom-binant protein, mainly present in membrane fractions
(Fig 3A), was purified by nickel-affinity chromatogra-phy from membrane protein extracts Following elu-tion with an imidazole gradient, collected fracelu-tions were loaded on a SDS⁄ PAGE gel (Fig 3A) A single
Fig 1 Phylogenetic analysis of bacterial, plant, fungal and protist NDH-2-like protein sequences Because lipoamide dehydrogenase shares a probable common ancestry with NDH-2 (30), the E coli sequence (LpdA) was chosen as a common root Corresponding GenBank (GenPept) accession numbers, NCBI RefSeq numbers or references of the protein sequences used are: E coli-LpdA, NP_308147; Acidianus ambiva-lens, CAD33806; C reinhardtii (N2Cr347); Arabidopsis thaliana (AtNDC), NP_568205; Synechocystis (slr1743), NP_441103; Nostoc-5, BAB75793; Synechocystis (sll1484), NP_442910; Nostoc-6, BAB76910; Leptospira interrogans, NP_714580; Chlorobium tepidum, NP_661273; Xanthomonas axonopodis, AAM38664.1; Brucella melitensis, AAL54028; A tumefaciens (AtuNDH-2), AI2824; Sinorhizobium meliloti, NP_386185; Mesorhizobium loti, NP_102176; Bradyrhizobium japonicum-1, NP_767691.1; Synechocystis (slr0851), NP_441107; Nostoc-1, BAB73083; Corynebacterium glutamicum, CAB41413; Mycobacterium smegmatis, AAC46302.1; Nostoc-4, BAB74663; Desulfovibrio desulfuricans, ZP_00130145; Cytophaga hutchinsonii, ZP_00309856; Bacteroides thetiaotaomicron, NP_810450; Neurospora crassa (NcNDI1), EAA27430; Trypanosoma brucei, AAM95239; S tuberosum (StNDA1), CAB52796; A thaliana (AtNDA1), NP_563783; N crassa (NcNDE1), CAB41986; S tuberosum (StNDB1), CAB52797; A thaliana (AtNDB1), NP_567801; C reinhardtii (N2Cr147), C reinhardtii (N2Cr247), S cerevisiae (NDI1), NP_013586; S cerevisiae (NDE1), NP_013865; S cerevisiae (NDE2), NP_010198; Yarrowia lipolytica (YlNDH-2), XP_505856; N crassa3, EAA29772; Burkholderia cepacia-2, ZP_00224966; Z mobilis, AAD56918; E coli, NP_415627; Haemophilus influenzae, NP_438906; B cepacia-1, ZP_00223855; A vinelandi, AAK19737; Pseudomonas fluorescens, AAF97237; B japonicum-2, NP_770367.1; Rhodopseudomonas palustris, ZP00010689; Halobacterium, NP_279851; B subtilis, NP_389111; Deinococcus radiodurans, NP_294674 When homologue sequences originating from the same organism were closely related, a single repre-sentative has been selected (e.g A thaliana NDAs or NDBs).
Trang 4protein band was eluted by 200 mm imidazole This
band, located below the 50 kDa molecular mass
marker (AtuNDH-2 has an expected size of 46 kDa),
was recognized by an antibody directed against the
poly(His) tag sequence and was recovered mainly in
the membrane fraction The purified enzyme was used
for further biochemical characterizations Following
heat denaturation of the enzyme, the flavine cofactor
was analysed by HPLC and identified as FAD
(Fig 3B) Figure 3C shows the absorption spectrum
of AtuNDH-2 protein Typical peaks of
FAD-con-taining proteins were observed, confirming the nature
of the cofactor [19] By using the extinction
coeffi-cient at 450 nm we calculated that the stoichiometric
ratio of flavinic cofactor per mole of protein reaches
1.02 mol, which is characteristic of NDH-2 proteins
[21] The capacity of the purified enzyme to oxidize
NADH or NADPH was measured by monitoring
absorbance decay at 340 nm in the presence of
var-ious electron acceptors (Table 1) In the presence of
ferricyanide, the NADH-oxidizing activity saturated
around 50 lm NADH and reached 100 nmolÆmin)1Ælg)1
protein In the presence of NADPH, a significant activity was measured, but it was not possible to observe saturation within a concentration range suit-able for spectrophotometric studies An activity
of 50 nmolÆmin)1Ælg)1 protein was measured at
200 lm NADPH Various quinone acceptors were tes-ted In the presence of the soluble quinone Q0, a NADH oxidation activity of 140 nmolÆmin)1Ælg)1 pro-tein was measured We also tested the ability of the protein to use decyl-ubiquinone (UQ) and decyl-PQ
as acceptors Because these quinones are poorly sol-uble in aqueous solutions, the rates measured in these experiments should be considered as indicative Never-theless, it clearly appears that AtuNDH-2 is able to catalyse the oxidation of NADH in the presence of both quinone acceptors (Table 1)
In the absence of acceptors, the enzyme was found
to oxidize NADH and NADPH at limited rates, prob-ably due to the capacity of the enzyme to interact directly with O2 [30] This property was studied by measuring O2 consumption using an O2 electrode (Table 2) In this assay, the affinity of the enzyme for
Fig 2 Conserved sequence motifs in NDH-2s from various organisms GenBank accession numbers: C glutamicum, CAB41413; S tubero-sum (StNDB1), CAB52797; S cerevisiae (NDE1), NP_013865; Synechocystis (slr1743), NP_441103 In the consensus sequence, conserved residues in at least five of seven or six sequences are indicated in one letter code: upper case when conserved in every sequence, lower case in other cases Functionally similar residues are marked with the following symbols: D, hydrophobic; fi , aromatic; #, acidic or neutral counterpart Grey shaded regions in C-terminal sequences represent hydrophobic fragments detected using MITOPROT II software (v 1.0) corresponding to the maximal local hydrophobicity indicated.
Trang 5NADPH was about four times lower than for NADH.
Similar Vmax values were measured for NADPH and
NADH, but under these conditions, measured Vmax
values were ~ 50 times lower than in the presence of
ferricyanide or quinone acceptors
In vitro interaction of AtuNDH-2 with bacterial and thylakoid membranes
The ability of AtuNDH-2 to interact with bacterial membranes was studied using membrane preparations
of an E coli mutant lacking both NDH-1 and NDH-2 The functionality of the respiratory chain of these preparations was first checked in the presence of succi-nate as substrate of complex II (data not shown) As expected, no O2 uptake was detected when mutant membranes were supplemented with NADH (Fig 4A) Preincubation of mutant membranes with AtuNDH-2 resulted in an O2 uptake upon NADH addition, whereas the enzyme alone consumed O2, but at a much lower rate (Fig 4B) At variance with respiratory O2 uptake, which leads to reduction of O2 into H2O, direct NADH oxidation activity of NDH-2 generates reactive oxygen species (ROS) [9] Superoxide dismu-tase (SOD) and catalase were added to the assay med-ium to convert back ROS into O2 and therefore minimize the contribution of direct oxidation which is not connected to electron transfer into the respiratory chain to O2 uptake Then, when assays on E coli membranes are performed in the presence of these scavengers, O2 uptake rates essentially reflect the qui-none reductase activity of the enzyme (Fig 4A,B) Under these conditions, at pH 7.2, similar Vmaxvalues (~3.7 nmol O2Æmin)1Ælg)1 protein) were obtained for both NADH and NADPH oxidations (data not shown) Diphenyleneiodonium (DPI), an inhibitor of flavin enzymes, inhibited both NADH- and NADPH-dependent reactions by ~75%, half inhibition being obtained at DPI concentrations of ~13 lm (data not shown)
A
B
C
Fig 3 Purification of His-tagged AtuNDH-2 by nickel-affinity
chro-matography (A) and analysis of the flavinic cofactor (B) (A)
Coo-massie Brilliant Blue-stained SDS⁄ PAGE and western blot analysis
using an anti-histidine IgG T, total protein extract from bacterial
cells expressing AtuNDH-2; S, soluble proteins; M, membrane
pro-teins solubilized by dodecyl maltoside; E1 and E2, eluted fractions
from nickel-affinity chromatography using 200 m M imidazole MW,
molecular mass markers (B) HPLC separation and fluorometric
ana-lysis of the flavinic cofactor Continuous line, cofactor extracted
from purified recombinant AtuNDH-2; dashed black line, FAD
stand-ard; dashed grey line, FMN standard (C) UV-visible spectrum of
0.3 l M of purified AtuNDH-2.
Table 1 Kinetic parameters of NADH oxidation determined on puri-fied AtuNDH-2 in the presence of various electron acceptors, using spectrophotometric measurements at 340 nm.
K m (l M ) V max (nmolÆmin)1Ælg)1protein) Ferricyanide (1 m M ) 5 100
Table 2 K m and V max of O 2 reduction by AtuNDH-2 measured using an O 2 electrode in the presence of NADH and NADPH as electron donors.
Km(l M ) Vmax(nmolÆmin)1Ælg)1protein)
Trang 6The ability of AtuNDH-2 to interact with PQs of
the photosynthetic electron transport chain was
stud-ied by performing chlorophyll fluorescence
measure-ments on thylakoid membranes of C reinhardtii
(Fig 5) When measured under nonactinic light, the
chlorophyll fluorescence level is an indicator of the
PQ pool redox state in the dark [31] Under anaerobic
conditions to prevent dark reoxidation of the PQ
pool, addition of NADH (200 lm) to C reinhardtii
thylakoid membranes provoked a slow increase in the
chlorophyll fluorescence level, indicating a reduction
of the PQ pool (Fig 5) This activity was recently
sug-gested to result from the activity of an endogenous
NDH-2-type enzyme [26] When AtuNDH-2 was
incu-bated with thylakoid membranes prior to chlorophyll
fluorescence measurements, the redox state of PQ
increased significantly more rapidly, the effect being
dependent on AtuNDH-2 concentrations (Fig 5)
AtuNDH-2 was also found to stimulate (two- to
threefold) light-dependent O2 uptake measured in
C reinhardtii thylakoid membranes in the presence
of NADH, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea
(DCMU) and methyl viologen (data not shown), this
measurement supplying an estimation of the electron
flow from NADH to PS I through the PQ pool [26]
We conclude from these experiments that AtuNDH-2
is able to interact with thylakoid membranes and reduce PQs
Functional complementation of a E coli mutant strain
Finally, we tested the ability of AtuNDH-2 to function
as a NADH dehydrogenase in vivo by studying its abil-ity to restore growth of an E coli mutant strain ANN0222 lacking both NDH-1 and NDH-2 Such a deleted strain has been reported to grow normally on a Luria–Bertani medium, but not be able to grow on a minimal medium supplemented with mannitol as the sole source of carbon [20] As shown in Fig 6A, con-trol transformants (corresponding to cells transformed with empty vector) could not grow on the minimal medium, nor could untransformed cells (data not shown) In contrast, the mutant strain expressing AtuNDH-2 after induction with 0.1 mm isopropyl thio-b-d-galactoside (IPTG) could grow under these condi-tions Note that partial complementation of the mutant was observed in the absence of IPTG, likely indicating significant IPTG-independent expression of the protein Using an antibody raised against the recombinant pro-tein, AtuNDH-2 was detected in protein extracts from the complemented ANN0222 strain (Fig 6C) Most of the protein was present in membrane fractions,
A
B C
Fig 5 Interaction of AtuNDH-2 with C reinhardtii thylakoid mem-branes The increase in chlorophyll fluorescence was measured under low light in response to NADH addition (final concentration
200 l M ) to a suspension of C reinhardtii thylakoids (30 lg chloro-phyllÆmL)1) (A) control; (B, C) thylakoid membranes were preincu-bated with purified AtuNDH-2 at two protein concentrations (2.5 and 5 lgÆmL)1final protein concentration, respectively) for 30 min before measurements.
A
B
Fig 4 Interaction of AtuNDH-2 with membranes of the E coli
mutant ANN0222 and effect of DPI (A) Effect of AtuNDH-2 on
NADH-dependent O2uptake measured in bacterial membranes
fol-lowing addition of 200 l M NADH; (a) control; (b) purified AtuNDH-2
(1.5 lgÆmL)1) was preincubated with bacterial membranes prior to
measurements; (c) effect of SOD (500 unitsÆmL)1) and catalase
(1000 unitsÆmL)1) addition to (b); (B) NADH-dependent O 2 reduction
by AtuNDH-2 (a) and effect of SOD and catalase addition (b).
Trang 7only a small portion being found in soluble proteins.
Significant expression of AtuNDH-2 was detected in
the absence of IPTG, consistent with the partial
com-plementation observed under these conditions
AtuNDH-2 activity was assessed on membrane
frac-tions prepared from the transformed mutant strain by
measuring NADH- and NADPH-dependent O2
con-sumption rates Whereas only low O2 uptake activity
was induced by NADH addition on membranes of the
control transformant strain, strong O2 uptake was
observed in membranes containing AtuNDH-2
(Fig 6B) Oxygen-uptake activities were measured in
membranes of the complemented ANN0222 strain to
determine apparent kinetic parameters of AtuNDH-2
Under these conditions, AtuNDH-2 oxidized both
NADH and NADPH with similar maximal rates, and
with a much higher affinity for NADH (Km¼ 12.5 lm)
than for NADPH (Km¼ 1129 lm) (data not shown)
Discussion
We report here the cloning, expression and characteri-zation of AtuNDH-2, the A tumefaciens orthologue to rotenone-insensitive NAD(P)H dehydrogenases The purified enzyme showed a NADH : Q0 oxidoreductase activity in the range of activities measured for other purified enzymes, such as the C glutamicum NDH-2 [9] or the Trypanosoma brucei NDH-2 [14] AtuNDH-2 appears, however, 1000 less active than the purified His-tagged E coli enzyme, the high activity of which has been attributed either to differences in purification protocols or to the preincubation with phospholipids [27] Like several other organisms, including E coli, Synechocystis PCC6803 and plant mitochondria, A tu-mefaciens appears to contain both a NDH-1 complex and a functional NDH-2 Whether AtuNDH-2 fulfils a bioenergetic function or acts as a sensor, as suggested
C
Fig 6 Growth complementation by AtuNDH-2 of a E coli mutant lacking both NDH-1 and NDH-2 (A) Colony forming assays on rich med-ium (Luria–Bertani) and minimal medmed-ium (M9) supplemented with mannitol Control, transformation control of the E coli strain ANN0222; AtuNDH-2, ANN0222 transformant strain expressing AtuNDH-2 under the control of an inducible promotor Induction was realized by addi-tion of 0.1 m M IPTG (B) O2uptake measurements in membranes of the E coli strain ANN0222 (control) and in the transformant strain expressing AtuNDH-2 NADH (200 m M final concentration) was added when indicated (C) Western blot analysis using an antibody raised against AtuNDH-2 S, soluble protein fraction; M, membrane protein fraction AtuNDH-2 accumulation was analysed in response to induction
by 0.1 or 0.5 m M IPTG Gel loading was 2.5 and 30 lg proteins in M and S lanes, respectively Right, immunodetection of purified AtuNDH-2, at 0.1, 0.2 and 0.3 lg protein ⁄ lane from left to right.
Trang 8in cyanobacteria [20], will require further study From
amperometric and chlorophyll fluorescence
measure-ments performed on E coli membranes and on C
rein-hardtii thylakoids, AtuNDH-2 is concluded to interact
with bacterial membranes and UQs and also with
thyl-akoid membranes and PQs Although NDH-2s are
membrane-bound enzymes, the nature of membrane⁄
protein interactions has not been elucidated
Mem-brane binding seems to rely on different mechanisms
depending on the enzyme, some involving
transmem-brane anchorage, whereas others involve electrostatic
interactions A membrane association via
amphipha-tic helices has been suggested for E coli and
Acidianus ambivalens NDH-2s [13,29] 2D structure
analysis, performed on the 50 NDH-2 sequences used
to build the phylogenetic tree, predict the presence of
C-terminal transmembrane helices in the prokaryotic B
subgroup (including AtuNDH-2) AtuNDH-2 also
contains a hydrophobic domain enriched in aromatic
residues located between its two cofactor binding sites
This domain has been proposed to be involved in the
interaction with hydrophobic quinones of respiratory
chains [11,13] Respective contributions of both
hydro-phobic domains to membrane and quinone interactions
require further study
In higher plants, addition of NAD(P)H to thylakoid
membrane preparations has been shown to stimulate
nonphotochemical reduction of the PQ pool [31]
Although higher plant chloroplasts contain a
func-tional NDH-1 complex [23,24], pharmacological
stud-ies concluded that a NDH-2-like enzyme is also
involved in this phenomenon [31] C reinhardtii
chlo-roplasts are recognized to lack NDH-1 complex [22]
A recent study conducted in our laboratory concluded
that, in C reinhardtii, the enzyme involved in the dark
reduction of PQs is a NDH-2-type enzyme [26]
Non-photochemical reduction of PQs is a reaction of
spe-cial interest because it may lead to the production of
hydrogen Indeed, under anaerobic conditions, C
rein-hardtii is able to efficiently produce hydrogen, using
solar energy and water, because of the existence of a
reversible hydrogenase connected to its photosynthetic
electron transport chain [32] Photobiological
hydro-gen production is a natural phenomenon of high
bio-technological interest [33,34], but is strongly limited by
the extreme O2 sensitivity of the hydrogenase, O2
being produced by PS II during photosynthesis To
overcome this limitation, the development of a
sequen-tial inhibition of PS II based on sulfur deprivation,
has been proposed [35] Under conditions of sulfur
deficiency, PS II activity is inhibited, allowing the
establishment of anaerobic conditions favourable to
hydrogen production In the absence of PS II,
hydro-gen production is possible thanks to the existence of a nonphotochemical PQs reduction pathway using stro-mal reductants originating from starch catabolism [36,37] This pathway, which allows hydrogen produc-tion without simultaneous O2 production and can sus-tain hydrogen production on a relatively long time scale [37], was recently proposed to involve a plastidial NDH-2 [26] Because nonphotochemical reduction of
PQ may constitute a limiting step of hydrogen produc-tion under certain experimental condiproduc-tions, overexpres-sion of a NDH-2 in C reinhardtii plastid should be considered as a valuable optimization strategy towards improving the anaerobic phase of hydrogen produc-tion AtuNDH-2 appears as a suitable gene for such a purpose because its high GC content is similar to that
of C reinhardtii genomic DNA The obtention of transformants expressing AtuNDH-2 is currently in progress in our laboratory
AtuNDH-2 showed a much higher affinity for NADH than for NADPH, although both substrates were oxidized with comparable Vmax values (Table 2) Several NDH-2s are strict NADH- [14,27] or strict NADPH-dehydrogenases [15,38], whereas a few others, mainly from plants, are able to oxidize both substrates indifferently [5] Michalecka et al [38] suggested that the presence of an acidic residue (E or D) at the end
of the second b sheet of the dinucleotide-binding site would confer NADH specificity by providing hydrogen bonding to the ribose moiety (Fig 2) In enzymes specifically oxidizing NADPH, this acidic residue is replaced by a neutral counterpart (Q or N) The pres-ence of a glutamic residue in AtuNDH-2 is in agree-ment with the enzyme preference for NADH However, AtuNDH-2 was able to oxidize NADPH at
a similar maximal rate, but with a lower affinity Although molecular basis of the NDH-2s ability to use both substrates are currently unknown, this property will be important for optimizing hydrogen production through chloroplast engineering, NADPH being gener-ally thought to be the major form of reducing power
in this compartment
Like most reported alternative NADH dehydrogen-ases [39,40], AtuNDH-2 was shown to contain FAD
as a prosthetic group FAD-containing enzymes dri-ving two-electron reduction are believed to be less likely than FMN-containing enzymes to produce su-peroxides [41] Nevertheless, AtuNDH-2, which con-tains FAD, has the ability to produce ROS at a significant rate (Table 1) Whereas very few other stud-ies have considered this autoxidation trait [9], Messner
& Imlay [30] have shown that E coli NDH-2 is a pri-mary site of superoxide formation in the aerobic res-piratory chain, with intensity varying according to its
Trang 9expression level and reduction status Such a property
may limit the potential biotechnological use of
NDH-2-like enzymes Expression of AtuNDH-2 in plastids
may therefore lead to the generation of additional
ROS which may alter cell viability and in the case
chloroplast-detoxifying mechanisms would be limiting
This property should be taken into consideration
in experiments aiming to overexpress NDH-2-like
enzymes in plastids
Experimental procedures
Strains and media
A tumefaciens C58 was grown in Luria–Bertani medium
(1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) in
the presence of rifampicine (100 lgÆmL)1) at 30C for 24 h
before genomic DNA extraction Cloning and gene
expres-sion were performed in E coli dH10b The E coli mutant
lacking both NDH-1 and NDH-2 ANN0222 was
gener-ously provided by T Friedrich (Freiburg University,
Germany) Complementation assays were performed on a
minimal medium supplemented with mannitol (1· M9 salts,
2· 10)3mMgSO4, 10)4mCaCl2, 0.4% mannitol)
Trans-formed bacteria were selected on Luria–Bertani medium
containing ampicillin (100 lgÆmL)1) C reinhardtii
wild-type (137c) was grown in a Tris-acetate phosphate medium
as described previously [42] Algal culture was maintained
at 25C under continuous agitation and under an
illumin-ation of 100 lmolÆphotonÆm)2Æs)1
Chemicals
All chemicals were purchased from Sigma-Aldrich (St
Louis, MO, USA) except DNP-INT which was generously
provided by A Trebst (Bochum University, Germany)
Cloning, expression and purification of
His-tagged NDH-2
The AtuNDH-2 gene was amplified from A tumefaciens
ge-nomic DNA, using the following couple of primers: (F)
CGCCAATTGATGCAAGAACATCATGTT; (R) AAAA
CTGCAGTCAATGATGATGATGATGATGGGCCTCG
TCCTTCAGCG MfeI and PstI sites were inserted in
for-ward and reverse primers, respectively, upstream and
down-stream the start and the stop codons, whereas a (His)6
-coding sequence was inserted in the reverse primer right
upstream the stop codon The amplified DNA was digested
by MfeI and PstI and ligated into the ampicillin-resistant
expression vector pSD80 [43], which was digested by EcoRI
and PstI, and introduced by electroporation in E coli
dH10b Ampicillin-resistant transformants were screened by
PCR for the presence of the AtuNDH-2 gene, using (F) and
(R) primers The construct has been verified by sequencing The plasmid was named pSDN2Ag6H and used to cotrans-form E coli dH10b with chloramphenicol-resistant pRare plasmid, carrying genes coding for the six rarest tRNA
of E coli (Merck Biosciences, Darmstadt, Germany) His-tagged NDH-2 was expressed in Luria–Bertani medium
in the presence of 50 lgÆmL)1 ampicillin and 25 lgÆmL)1 chloramphenicol and incubated at 37C under vigorous shaking Expression was induced for 2 h by the addition of 0.5 mm IPTG when the culture reached D¼ 0.5 Cells were then harvested by centrifugation and washed with a solu-tion containing 500 mm KCl, 10 mm Tris⁄ HCl, pH 7.5, 0.2 mm phenylmethylsulfonyl fluoride and stored at )80 C
Protein purification Membrane isolation and nickel-affinity purification of
A tumefaciens His-tagged NDH-2 were performed follow-ing the protocol developed by Bjo¨rklo¨f et al [27] for purifi-cation of an His-tagged E coli NDH-2 Membranes were solubilized using dodecyl maltoside and the enzyme was purified by FPLC (A˚kta˚ FPLC, Amersham Biosciences, Uppsala, Sweden) using a HiTrap chelating nickel column (Amersham Biosciences) The bound NDH-2 was eluted using an imidazole gradient Collected fractions were ana-lysed by SDS⁄ PAGE
Immunological analysis
A commercial antibody directed against the His-tag sequence (Sigma ref H-1029) was used to identify the puri-fied protein The puripuri-fied enzyme was collected in a 5 mL volume and concentrated by ultrafiltration to a 900 lL final volume Glycerol 100% (200 lL) was added before )80 C storage The enzyme concentration was estimated by SDS⁄ PAGE comparing to a range of BSA standards A rabbit serum was raised against the purified AtuNDH-2 protein (Agro-Bio, Villeny, France) and further used to probe the presence of the AtuNDH-2 protein in soluble and membrane protein fractions Proteins were separated
on a 10% SDS⁄ PAGE gel and transferred to a nitrocellu-lose membrane using a semidry transfer technique The appropriate antibody was correctly diluted: 1 : 2000 diluted His IgG (Fig 3); 1 : 10 000 diluted polyclonal anti-(AtuNDH-2) serum (Fig 6) For the anti-His IgG, the detection reaction (anti-rabbit alkaline phosphatase conju-gated as secondary antibody) was performed according to the protocol recommended by the manufacturer (Sigma) For the anti-(AtuNdh2) IgG, the Alexa 680 goat anti-rabbit (Invitrogen, Molecular Probes, Carlsbad, CA, USA) was used as secondary antibody and the detection was per-formed by using the LICOR (Lincoln, NE, USA) Odyssey system
Trang 10Colony-forming assays
ANN0222 competent cells were transformed either with
pSDN2ag6 h plasmid or empty vector as a control and
transformants were selected on Luria–Bertani agar
contain-ing ampicillin Transformants were grown to
mid-exponen-tial phase at 37C in ampicillin-containing Luria–Bertani
medium Cells were washed once with sterile M9 medium
supplemented with mannitol as the sole source of carbon
Cells were then diluted in M9⁄ mannitol medium and
spot-ted on M9⁄ mannitol ⁄ agar plates containing ampicillin and
different IPTG concentrations Plates were incubated at
37C for two days As a control, cells were plated on a
Luria–Bertani agar medium containing ampicillin and
appropriate IPTG concentrations and incubated at 37C
overnight
Flavin analysis
The nature of the flavinic cofactor was determined as
des-cribed previously [14] The purified enzyme was boiled for
3–4 min followed by centrifugation Supernatant was
ana-lysed by HPLC using a 150· 4.6 mm internal diameter
Supelcosil LC-DP column (Sigma-Aldrich) The
composi-tion of the mobile phase (flow rate of 1 mLÆmin)1) was
80% of 0.1% TFA in water and 20% of 0.1% TFA in
40% acetonitrile Excitation and emission wavelengths of
the fluorescence detector were set at 450 and 525 nm,
respectively FAD and FMN standards were used to
iden-tify the nature of the flavinic cofactor UV-visible spectra
of native and boiled proteins were recorded using a
Cary-50 spectrophotometer (Varian, Palo Alto, CA, USA)
Pro-teins were diluted in 50 mm Tris⁄ HCl buffer pH 7.5,
10 mm NaCl, 5 mm MgCl2 and the FAD content was
determined spectrophotometrically in protein free
superna-tant from boiled samples (e450¼ 11 300 m)1Æcm)1)
Preparation of bacterial and thylakoid
membranes
A 50 mL Luria–Bertani–tetracyclin E coli ANN0222
cul-ture was harvested at the end of exponential growth phase
(D¼ 1) by centrifugation (15 min, 3200 g) Cells were
washed twice and resuspended in 10 mL of lysis buffer A
(200 mm Tris⁄ Cl pH 8, 2.5 mm EDTA and 0.2 mm
phenyl-methylsulfonyl fluoride) according to Bjo¨rklo¨f et al [27]
Cells were disrupted by passing twice through a chilled
French pressure cell maintained at 16 000 p.s.i., and
mem-brane fraction was collected by centrifugation (30 min,
4C, 48 500 g, Beckman JA-20 rotor; Beckman Coulter,
Fullerton, CA, USA) Membranes were resuspended in
200 lL of analysis buffer B (50 mm phosphate buffer,
pH 7.5 and 150 mm NaCl) according to Bjo¨rklo¨f et al [27]
The same procedure was followed to prepare membranes
from ANN.0222 transformed cells either with pSD80 or pSDN2Ag6H
A 200 mL volume of C reinhardtii culture grown on Tris-acetate phosphate medium was harvested in exponential growth phase, centrifuged, washed in 35 mm Hepes-NaOH buffer, pH 7.2 and resuspended in 12 mL of buffer C (50 mm Tricine-NaOH, 10 mm NaCl, 5 mm MgCl2; pH 8) supplemented with 1% BSA w⁄ v, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride The following operations were carried out in the dark at 4C Cells were disrupted by two cycles of a chilled French pressure cell (2000 p.s.i) The homogenate was centrifuged at 500 g for 5 min using
an Eppendorf 8510R centrifuge (Eppendorf, Hamburg, Germany) The pellet, containing unbroken cells, was discar-ded The supernatant was centrifuged at 3000 g for 15 min using an Eppendorf 8510R centrifuge to collect thylakoid membranes Thylakoid membranes were resuspended in 250–500 lL of buffer C and stored on ice, in the dark Chlo-rophyll extraction was performed in 80% acetone⁄ H2O v⁄ v, and chlorophyll concentration was calculated from absorp-tion measurements at 663 and 646 nm [44]
Spectrophotometric measurement of enzymatic activity
Assays of NAD(P)H oxidation were performed by monitor-ing absorbance decrease at 340 nm, concentration variations being deduced from these measurements by applying the extinction coefficient of NAD(P)H at this wavelength (6.22 mm)1Æcm)1) NAD(P)H, acceptors and protein extracts (incubated or not with membranes of E coli ANN0222) were added successively Bovine SOD (500 unitsÆmL)1) and cat-alase (1000 unitsÆmL)1) were added to the assay medium
O2uptake by bacterial and thylakoid membranes
O2uptake was measured using a Clark electrode (DW2⁄ 2, Hansatech, King’s Lynn, UK) In some experiments, a reaction mixture containing 10 lL of membranes of E coli ANN0222 and 1.5 lg of purified enzyme was preincubated
10 min on ice, then diluted in 990 lL of buffer B and intro-duced into the electrode chamber O2 consumption was measured at 25C in the presence of NADH or NADPH
O2uptake was also followed in absence of bacterial mem-branes to measure direct oxidation by the enzyme Catalase (1000 unitsÆmL)1) and SOD (500 unitsÆmL)1) were added to the assay medium Inhibitory effects of DPI were quantified after 10 min incubation with membrane samples before measurements
Chlorophyll fluorescence measurements Measurements were achieved at 25C, in anaerobic condi-tions a pulse modulated amplitude fluorometer (PAM