YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR.. Analysis of the form of Moco present in YagTSR reve
Trang 1molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli
Meina Neumann1, Gerd Mittelsta¨dt1, Chantal Iobbi-Nivol2, Miguel Saggu3, Friedhelm Lendzian3, Peter Hildebrandt3 and Silke Leimku¨hler1
1 Institute of Biochemistry and Biology, University of Potsdam, Germany
2 Laboratoire de Chimie Bacterie`nne, IFR 88 Institut Biologie Structurale et Microbiologie, CNRS, Marseille, France
3 Max-Volmer-Laboratories, Institut fu¨r Chemie, Technische Universita¨t Berlin, Germany
Molybdoenzymes are involved in a large number of
enzymatic reactions in the nitrogen, carbon and sulfur
cycles They occur in all the kingdoms of life With the
exception of nitrogenase, all molybdoenzymes carry
the molybdenum cofactor (Moco), where the
molybde-num atom is coordinated to the unique dithiolene
moiety of a conserved tricyclic pyranopterin cofactor
called molybdopterin (MPT) Depending on the remaining ligands of the molybdenum center, molyb-doenzymes are classified into three families: (a) the xanthine oxidase (XO) family, characterized by a cyanolyzable equatorial sulfur ligand coordinated to the molybdenum atom; (b) the sulfite oxidase family, with two oxo ligands at the molybdenum center; and
Keywords
aldehyde oxidoreductase;
aromatic aldehyde; MCD; molybdenum;
molybdo-flavoenzyme
Correspondence
S Leimku¨hler, Institute of Biochemistry
and Biology, University of Potsdam,
D-14476 Potsdam, Germany
Fax: +49 331 977 5128
Tel: +49 331 977 5603
E-mail: sleim@uni-potsdam.de
(Received 2 February 2009, revised 10
March 2009, accepted 11 March 2009)
doi:10.1111/j.1742-4658.2009.07000.x
Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron– sulfur flavoprotein from E coli, which is located in the periplasm The
135 kDa enzyme comprised a noncovalent (abc) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a med-ium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa)
2· [2Fe2S]-containing YagT subunit YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR YagTSR represents the first example of a molyb-dopterin cytosine dinucleotide-containing protein in E coli Kinetic charac-terization of the enzyme revealed that YagTSR converts a broad spectrum
of aldehydes, with a preference for aromatic aldehydes Ferredoxin instead
of NAD+ or molecular oxygen was used as terminal electron acceptor Complete growth inhibition of E coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E coli under certain growth conditions
Abbreviations
ICP-OES, inductively coupled plasma optical emission spectroscopy; MCD, molybdopterin cytosine dinucleotide; MGD, molybdopterin guanine dinucleotide; Moco, molybdenum cofactor; MPT, molybdopterin; Tat, twin arginine protein transport; XDH, xanthine dehydrogenase;
XO, xanthine oxidase.
Trang 2(c) the dimethylsulfoxide reductase family, where one
molybdenum atom is coordinated by two dithiolene
groups [1–3] Whereas in eukaryotes Moco is present
solely in its Mo-MPT form, in prokaryotes Moco can
be further modified by the addition of
mononucleo-tides such as GMP, CMP, IMP or AMP to the
phosphate group of the MPT [4–7] In general, Moco
formed in Escherichia coli is further modified by
covalent addition of GMP to the phosphate atom on
C4¢ of MPT via a pyrophosphate bond, a reaction
catalyzed by the mobAB gene products Furthermore,
two of the molybdopterin guanine dinucleotide
(MGD) moieties are ligated to the molybdenum atom
via the dithiolene group of MPT, forming bis-MGD
So far, only the YedY protein has been shown to bind
the Mo-MPT form of Moco in E coli; however, the
physiological role of this protein still remains unclear
[8] In general, prokaryotic members of the XO family
have been shown to bind the molybdopterin cytosine
dinucleotide (MCD) form of Moco, containing CMP
added to the terminal phosphate group of the pterin
side chain [9]
The XO family of molybdoenzymes comprises a
number of different enzymes in prokaryotes and
eukaryotes, transferring oxygen derived from water to
their substrate Most enzymes of the XO family are
well characterized as purine-oxidizing and⁄ or
alde-hyde-oxidizing enzymes with broad substrate
specifici-ties, but several more specific enzymes, such as carbon
monoxide dehydrogenase and nicotine dehydrogenase,
have been described [10,11] Well-characterized
enzymes with aldehyde-oxidizing activity are
Desulfo-vibrio gigas aldehyde oxidoreductase and mammalian
aldehyde oxidases [12] Mammalian aldehyde oxidases
are expressed at high levels in the liver and in the lung,
and have been implicated in the detoxification of
envi-ronmental pollutants and xenobiotics [13] Bacterial
aldehyde oxidases and aldehyde dehydrogenases have
been identified in different bacteria, including
Methylo-coccus sp., Pseudomonas sp., Streptomyces moderatus
[14], Amycolatopsis methanolica [15], and
Pseudo-monas testosteroni[16] In addition, xanthine
dehydro-genases (XDHs) capable of oxidizing various purine
and aldehyde substrates have been characterized in
bacteria such as Rhodobacter capsulatus [17],
Pseudo-monas putida86 [18,19], and Veillonella atypica [20,21]
With the exception of R capsulatus and
Pseudomo-nas aeruginosaXDH [22,23], which bind Mo-MPT, all
bacterial XDHs characterized to date bind the MCD
form of Moco The molecular masses of these XDHs
range from 140 to 300 kDa, and different subunit
structures have been observed, such as a2 in
Strepto-myces cyanogenus [24], abc in V atypica [21], a3 in
P putida[25], a2b2in R capsulatus [17], a2b2 in Coma-monas acidovorans [26,27], and a4b4 in P putida 86 [19] However, in general, enzymes of the XO family possess the same overall architecture [28], with two dis-tinct [2Fe2S] clusters bound to the N-terminal domain
or subunit, an FAD bound to a central domain or subunit (with the exception of D gigas aldehyde oxi-doreductase, in which the FAD-binding domain is absent [29]), and the Moco-binding domain at the C-terminus
As part of the E coli K-12 genome project [30], three DNA regions carrying genes encoding putative homologs of XDHs were identified, named xdhABC, xdhD, and yagTSRQ [17,31]; however, none of these proteins has been characterized at the biochemical level
to date, and their physiological functions remain as yet unknown Previous amino acid sequence alignments with the individual domains of the well-characterized bacterial XDH from R capsulatus revealed amino acid identities of 24–43% between single protein domains; however, the organization of the genes was found to
be different to that in R capsulatus xdhA, xdhB and xdhC and their putative E coli counterparts [17] Alignments of two of the annotated operons in E coli, xdhABC and xdhD, showed higher homologies to XDHs than to aldehyde oxidases [17] Genetic approaches suggested a role in the purine salvage path-way for these enzymes [31] The third operon, yagTSRQ, encodes a putative aldehyde oxidase In the yagTSRQ operon, YagT contains a twin arginine pro-tein transport (Tat) leader peptide for translocation to the periplasm Reporter protein fusion assays revealed that the YagT signal peptide leads to export to the periplasm and cleavage after amino acid 49, so YagTSRQ was predicted to contain a periplasmic protein complex [32] YagT shares homologies with the FeS cluster-containing subunit of the class of molyb-do-flavoenzymes, YagS with the FAD-binding subunit, and YagR with the Moco-containing subunit [17] YagQ was shown to share amino acid homology of 40% to R capsulatus XdhC, a protein that has been shown to be involved in Moco binding, addition of the terminal sulfido ligand of Moco, and insertion of sulfurated Moco into the XdhB subunit of R capsula-tus XDH [33] The yagTSRQ operon is not essential for E coli under standard growth conditions, as a gene region containing the yagTSRQ operon is deleted in the E coli K-12-based laboratory strain MC4100 and its derivatives [34] As the yagTSRQ operon contains
an ORF for a protein homologous to R capsulatus XdhC, and is the least characterized operon at the genetic level of members of the XO family, we analyzed the role of the yagTSRQ operon in E coli
Trang 3In the present work, we describe the purification and
characterization of gene products of the yagTSRQ
operon in addition to the characterization of their
physiological role in E coli YagTSR was shown to be
a periplasmic aldehyde oxidoreductase that oxidizes a
broad spectrum of aldehydes As complete growth
inhibition of E coli cells devoid of genes from the
yagTSRQ operon was observed by the addition of
cinnamaldehyde to a low-pH medium, we suggest that
a periplasmic aldehyde oxidoreductase might play a
role in the detoxification of aldehydes to avoid cell
damage in E coli
Results
Purification of YagTSR expressed in the presence
or absence of YagQ
YagTSRQ and YagTSR were expressed in E coli
TP1000 (DmobAB) cells from plasmids pMN100 and
pMN111, respectively Expression of the proteins results
in an N-terminal His6-fusion to YagT, with a deletion
of the 49 N-terminal amino acids of the Tat leader
peptide TP1000 (DmobAB) is a derivative of the E coli
MC4100 strain [35], which carries a deletion of the gene region encompassing the yagTSRQ operon Thus, no endogenous yagQ was present to interfere with our analyses After purification by Ni2+–nitrilotriacetic acid affinity chromatography, eluted fractions from the two different expression constructs displayed three bands on Coomassie Brilliant Blue R-stained SDS⁄ PAGE gels, corresponding to molecular masses of 78.1, 33.9 and 21.0 kDa, respectively (Fig 1A) Coex-pressed yagQ was not identified as a subunit of the purified YagTSR enzyme expressed from pMN100 Densitometric analysis of the Coomassie-stained SDS⁄ PAGE gel revealed comparable peak densities for the YagT and YagS subunits from both expressions; however, the peak density of YagR was reduced to 37% in the strain containing YagTSR expressed in the absence of YagQ (Fig 1A) The protein expressed from the yagTSRQ operon eluted with a size of 135 kDa from a Superdex 200 size exclusion chromatography column, corresponding to the YagTSR trimer (Fig 1B, solid line) The protein expressed in the absence of YagQ ()YagQ) displayed two peaks after Superdex 200 size exclusion chromatography, the 135 kDa peak cor-responding to the YagTSR trimer, and a 55 kDa peak
Fig 1 Purification of YagTSR after expression in the presence and absence of YagQ (A) 12% SDS ⁄ PAGE of purification stages Lane I:
1 lL of E coli TP1000 · pMN100 (yagTSRQ) extract after cell lysis Lane II: 10 lL of YagTSR with an OD 445 nm of 0.09 after expression in the presence of YagQ Lane III: 1 lL of E coli TP1000 · pMN111 (yagTSR) extract after cell lysis Lane II: 10 lL of YagTSR with an
OD 445 nm of 0.09 after expression in the absence of YagQ (B) Size exclusion chromatography of YagTSR Two hundred microliters of YagTSR (+YagQ, solid line, )YagQ, dotted line) with an OD 445 nm of 0.26 was analyzed by analytical size exclusion chromatography in
50 m M Tris and 200 m M NaCl (pH 7.5) using a Superdex 200 column YagTSR (+YagQ) was additionally purified on a Q-Sepharose column prior to size exclusion chromatography Inset: plot of the standard proteins Size exclusion chromatography markers (Bio-Rad): c-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12(1.3 kDa).
Trang 4containing to the YagTS dimer (Fig 1B, dotted line).
Analysis of the specific activities of both proteins with
vanillin revealed that YagTSR expressed in the absence
of YagQ ()YagQ) was completely inactive, whereas
YagTSR expressed in the presence of YagQ (+YagQ)
exhibited an activity of 27.9 ± 0.2 UÆmg)1(Table 1)
Cofactor analysis of the purified YagTSR proteins
To analyze the cofactor content of YagTSR expressed
in the presence or absence of YagQ, the molybdenum
and iron contents were quantified by inductively
cou-pled plasma optical emission spectroscopy (ICP-OES)
(Table 1) Iron contents of 4.04 ± 0.15 molecules per
YagTSR (+YagQ) and 3.84 ± 0.08 molecules per
YagTSR ()YagQ) were identified, corresponding to
two [2Fe2S] clusters per protein trimer The inactive
YagTSR ()YagQ) protein was shown to contain no
molybdenum bound to the protein, whereas the active
YagTSR (+YagQ) trimer was saturated to 58 ± 3%
with molybdenum
To analyze the Moco content of YagTSR (+YagQ)
and YagTSR ()YagQ), the proteins were incubated
for 30 min at 95C in the presence of acidic iodine,
which oxidizes released MPT to its fluorescent
deriva-tive, Form A Whereas Form A was readily detected
from YagTSR (+YagQ) after separation on a
reversed-phase C18 column (Fig 2A), no Form A was
released from YagTSR ()YagQ) (Fig 2B) In contrast,
after overnight incubation of YagTSR (+YagQ) with
acidic iodine at room temperature, and purification
using a Q-Sepharose column, no Form A was detected
(Fig 2C) Instead, a nucleotide derivative of Form A
was eluted from the Q-Sepharose column (Fig 2D)
As YagTSR (+YagQ) was purified from the E coli
TP1000 strain (DmobAB) in an active form, the
pres-ence of bis-MGD bound to YagTSR could be
excluded To identify the nucleotide bound to Moco in YagTSR (+YagQ), the protein was incubated for
15 min at 95C in the presence of 5% sulfuric acid, which released AMP from FAD and the nucleotide of
Table 1 Cofactor content of YagTSR expressed in the presence or absence of YagQ Specific enzyme activity (unitsÆmg)1) is defined as the oxidation of 1 lmol vanillinÆmin)1Æmg)1in phosphate ⁄ citrate buffer (pH 6.0) at room temperature, using ferricyanide as electron acceptor Molybdenum (l M molybdenum ⁄ l M YagTSR) and iron (l M 2 · [2Fe2S] ⁄ l M YagTSR) contents were determined by ICP-OES (see Experimental procedures) and related to a fully saturated enzyme Nucleotide content (l M CMP or AMP ⁄ l M YagTSR) content was analyzed after release
of CMP from MCD and AMP from FAD by heat treatment under acidic conditions, as described in Experimental procedures The concentra-tion of the terminal sulfur ligand of Moco (l M SCN)⁄ l M YagTSR) was determined spectrophotometrically as an iron–thiocyanate complex at
420 nm as described in Experimental procedures Potassium thiocyanate was used as a standard curve ND, none detectable; –, not deter-mined.
Expression strain Activity (unitsÆmg)1) Mo (%) Fe (%) AMP (%) CMP (%) Cyanolyzable sulfur (%)
a YagTSR expressed from plasmid pMN100 (yagTSRQ) was purified by Ni 2+ –nitrilotriacetic acid and Q-Sepharose chromatography as described in Experimental procedures.bYagTSR expressed from plasmid pMN111 (yagTSR) was purified solely by Ni2+–nitrilotriacetic acid chromatography as described in Experimental procedures.
Fig 2 Moco analysis of YagTSR expressed in the presence or absence of YagQ Analysis of the fluorescent derivatives of Moco from YagTSR Form A was produced from (A) 2 l M YagTSR (+YagQ) and (B) 2.4 l M YagTSR ( )YagQ) after 30 min of oxidation with acidic iodine at 95 C Form A was separated on a C18 RP-HPLC column with 85% 5 m M ammonium acetate and 15% methanol at an isocratic flow rate of 1 mLÆmin)1 (C) Form A was produced after overnight oxidation in acidic iodine at room tempera-ture Released Form A was applied to a Q-Sepharose column, eluted with 10 m M acetic acid, and applied to a C18 RP-HPLC column in 85% 5 m M ammonium acetate and 15% methanol at an isocratic flow rate of 1 mLÆmin)1 (D) The dinucleotide form of Form A was produced after overnight oxidation in acidic iodine at room temperature Released Form A dinucleotide was applied to a Q-Sepharose column, eluted with 50 m M HCl, and applied to a C18 RP-HPLC column in 97% 5 m M ammonium acetate and 3% metha-nol at an isocratic flow rate of 1 mLÆmin)1 Fluorescence was deter-mined by excitation at 383 nm and emission at 450 nm.
Trang 5the MPT dinucleotide cofactor Nucleotide analysis
using the respective standard nucleotides revealed the
presence of AMP and CMP (Table 1) The AMP
content could be related to complete saturation of
YagTSR (+YagQ) and YagTSR ()YagQ) with FAD
(Table 1) CMP was present in YagTSR (+YagQ) at a
saturation level of 62 ± 6% per trimer, but was not
identified in YagTSR ()YagQ) (Table 1) The CMP
content corresponded well to the molybdenum content
of YagTSR (+YagQ), and showed that the protein
was saturated to 60% with the MCD cofactor
As YagTSR belongs to the XO family of
molyb-doenzymes, characterized by a terminal sulfido ligand
at Moco, it was of interest to determine the saturation
of the sulfur ligand in YagTSR (+YagQ) As shown
in Table 1, after incubation of YagTSR (+YagQ) with
cyanide, the content of the cyanolyzable sulfur was
determined to be 58 ± 4% This result showed that
the MCD cofactor present in YagTSR (+YagQ) was
completely saturated with the sulfido ligand, and
no demolybdo or desulfo form of the protein was
purified
Steady-state kinetics of YagTSR, determined
using different aldehydes and purines as
substrates
For kinetic characterizations, YagTSR was purified
by Ni2+–nitrilotriacetic acid chromatography and
Q-Sepharose ion exchange chromatography (Fig 3A)
The yield of purified YagTSR was 1.8 mgÆL)1 of
E coli culture The visible absorption spectrum of
YagTSR (+YagQ) was similar to those of other
molybdo-flavoenzymes, and showed the presence of FeS and FAD as prosthetic groups (Fig 3B) Reduc-tion of YagTSR (+YagQ) with benzaldehyde showed that the protein was reduced to a level of 60% under anaerobic conditions For complete reduction, sodium dithionite was added to a final concentration of 20 mm (Fig 3B) During the reduction of YagTSR with either benzaldehyde or dithionite, the production of the flavin semiquinone was not observed
Steady-state kinetics with YagTSR showed a broad substrate spectrum with aromatic and aliphatic alde-hydes, whereas purines were not oxidized (Table 2) In total, aromatic aldehydes were converted with higher
kcatand lower Kmvalues than those for aliphatic alde-hydes The lowest Kmvalues of 69.8 and 63.1 lm were obtained for benzaldehyde and cinnamaldehyde, respectively Both substrates also showed the highest catalytic efficiency, with kcat⁄ Km ratios of 1.39 and 1.32 lm)1Æs)1, respectively In comparison, the Km of vanillin of 131.8 lm was about twice as high, with a concomitant increase in kcat to 124.6 s)1 Phenylacetic aldehyde showed the lowest kcat, of 7.0 s)1 None of the tested aliphatic aldehydes showed a Km below
400 lm, indicating that these are not likely to be physi-ological substrates of YagTSR Retinalaldehyde, an aromatic aldehyde with a long aliphatic side chain, was not oxidized by YagTSR As enzymes of the XO family are known to catalyze the conversion of a vari-ety of purines, xanthine, hypoxanthine and caffeine were additionally analyzed as substrates for YagTSR
In summary, no activity was detected with all purines tested during the steady-state kinetic analyses Additionally, YagTSR showed no detectable nicotine
A B
Fig 3 Purification and UV–visible absorption spectra of YagTSR (A) Twelve percent SDS ⁄ PAGE of purification stages of YagTSR (+YagQ) Lane I: molecular weight marker Lane II: 1 lL of E coli TP1000 · pMN100 (yagTSRQ) extract after cell lysis Lane III:
12 lg of YagTSR after Ni 2+ –nitrilotriacetic acid affinity chromatography Lane IV: 12 lg of YagTSR after Q-Sepharose ion exchange chromatography (B) Characterization of purified YagTSR (+YagQ) by UV–visible absorption spectroscopy Spectra of 7 l M air-oxidized YagTSR (solid line), of 7 l M YagTSR incubated with 500 l M benzaldehyde (dashed line), and of 7 l M YagTSR reduced with 20 m M dithionite (dotted line) Spectra were recorded in 50 m M Tris and 1 m M EDTA (pH 7.5) under anaerobic conditions.
Trang 6dehydrogenase activity Thus, YagTSR was identified
as an aldehyde-oxidizing enzyme To identify the
phys-iological electron acceptor, nonphysphys-iological electron
acceptors, such as 2,6-dichlorophenol-indophenol and
ferricyanide, in addition to cytochrome c, NAD+,
molecular oxygen or ferredoxin, were analyzed As
shown in Table 3, no activity was observed when
cyto-chrome c, NAD+or O2was used as terminal electron
acceptor Although the nonphysiological electron
acceptors dichlorophenol-indophenol and ferricyanide
were suitable electron acceptors, spinach ferredoxin
was also able to accept electrons from reduced
YagTSR This indicates that an E coli ferredoxin
might act as a physiological electron acceptor
(Table 3)
We additionally analyzed the pH optimum and the
temperature stability of the enzyme (Fig 4) YagTSR
showed a pKa of 5.5 with vanillin as substrate and
ferricyanide as electron acceptor (Fig 4A), revealing
the pKa of the active site glutamate (Glu692), which
acts as a base catalyst The exchange of Glu692 for
glutamine resulted in an inactive enzyme (the rate of
reaction was at least 107-fold slower than seen for the
wild-type enzyme; data not shown), underlining the
importance of this residue in the base-catalyzed
reac-tion The protein was stable up to a pH of 4; however,
at lower pH, the protein was denatured and release of
FAD was observed (data not shown) The effect of
temperature on the enzyme activity revealed that the
protein was heat stable at temperatures of up to 95C
in short-term incubations (data not shown) In long-term incubations at higher temperatures, YagTSR retained 92% of its activity after 15 min at 50C, and 73% of its activity after 15 min at 70C (Fig 4B)
EPR spectroscopy of the YagTSR FeS clusters Figure 5 shows the EPR spectra obtained from YagTSR at low temperatures; these were obtained after reduction of the samples with either benzaldehyde
or sodium dithionite Proteins from the XO family usually exhibit signals from two different [2Fe2S] clus-ters, which can be clearly distinguished by EPR spec-troscopy, owing to their different g-values and temperature behaviors [2,36,37] The signal showing more axial symmetry, which is also visible at higher temperatures (e.g 60 K), is assigned to the FeSI clus-ter, and the second signal, which is only visible at tem-peratures below 40 K, is assigned to the FeSII cluster
In YagTSR, a more rhombic signal at 2.005, 1.943 and 1.916 could be seen after sodium dithionite reduc-tion at a temperature of 60 K (for simulareduc-tion parame-ters, see Table 4) The gavwas 1.95 and, typically for a [2Fe2S] center, the linewidth was 1.4 mT This signal can be attributed to the FeSI cluster When the tem-perature was decreased to below 40 K, the signal of FeSII appeared to be superimposed on the FeSI signal (Fig 5B) The corresponding g-values of FeSII were
Table 3 Analysis of different electron acceptors for YagTSR Spe-cific enzyme activity (unitsÆmg)1) is defined as the oxidation of
1 lmol of vanillinÆmin)1Æmg)1 of enzyme in buffer containing
100 m M Tris (pH 6.8) ND, none detectable.
Electron acceptor Specific activity (UÆmg)1)
2,6-Dichlorophenol-indophenol f 2.73 ± 0.08
a Reduction of 0.2 mgÆmL)1spinach ferredoxin was determined by following the absorbance change at 420 nm. bReduction of 0.65 mgÆmL)1 cytochrome c was determined by following the absorbance change at 550 nm c Reduction of 1 m M NAD + was determined by following the reduction at 340 nm; 500 l M benzalde-hyde was used as substrate to avoid the overlap in absorption at
340 nm of vanillin d Activity was measured using either oxygen-saturated phosphate ⁄ citrate buffer (pH 6.0) or Tris buffer (pH 6.8)
by following the oxidation of vanillin at 340 nm.eReduction of
1 m M ferricyanide was determined by following the absorbance change at 420 nm f Reduction of 200 l M 2,6-dichlorophenol-indo-phenol was determined by following the absorbance change at
600 nm.
Table 2 Steady-state kinetics parameters of YagTSR with different
aldehyde and purine substrates Steady-state kinetics were
deter-mined in phosphate ⁄ citrate buffer (pH 6.0) using 1 m M ferricyanide
as electron acceptor at substrate concentrations of approximately
0.5–2 K m K m and k cat values were obtained after nonlinear fitting
using ORIGIN 6.0 software (Microcal; GE Healthcare) ND, none
detectable; –, not determined.
kcat⁄ K m
(l M )1Æs)1)
Phenylacetic aldehyde 132 ± 6 7.0 ± 0.6 0.05
2,4-Dihydroxybenzaldehyde 428 ± 55 55 ± 8 0.13
Trang 72.07, 1.96, and 1.92 The linewidth was 2.4 mT The
gavvalue of 1.98 is similar to that for FeSII from other organisms [38] The linewidth of FeSI at low tempera-tures was slightly increased, especially on the gx -com-ponent This broadening probably resulted from slight heterogeneity in the local structure of FeSI, which averaged out at 60 K Alternatively, it may have resulted from a coupling between FeSI and FeSII, which consequently is expected to lead to a small split-ting at low temperatures The linewidth of both clus-ters was smaller than, for example, that of the [2Fe2S] clusters from R capsulatus XDH [36]
In addition to reduction with sodium dithionite, the sample was treated with the substrate benzaldehyde (Fig 5C) The resultant spectrum was identical to the dithionite reduced spectrum (Fig 5B), showing that the FeS clusters were indeed reduced by electron trans-fer in the course of the catalytic reaction The spec-trum at higher temperatures, which only reflects FeSI, was identical to the spectrum in Fig 5A (data not shown)
The electron transfer seemed to be quite fast, and
no signal attributable to a flavin semiquinone radical could be found at pH 7.5 However, when reduction at
pH 10 was performed, the reaction was considerably slower, and the flavin semiquinone radical became visible (data not shown)
Growth of E coli wild-type and different mutant strains in the presence and absence
of cinnamaldehyde
To determine the physiological role of YagTSR in
E coli, yagR), yagT) and yagQ) cells were grown in 0.1· LB medium at pH 4.0 in the presence or absence
of cinnamaldehyde As shown in Fig 6A, in the absence of cinnamaldehyde the growth of the strains
A B
Fig 4 Analysis of the pH optimum and temperature stability of YagTSR (A) The pH optimum of YagTSR was determined by analysis of the specific activity (unitsÆmg)1)
in phosphate ⁄ citrate buffer in a pH range from 4 to 8, with ferricyanide as electron acceptor (B) For the analysis of the temper-ature stability of YagTSR, the enzyme was incubated for 15 min at different tempera-tures The specific cinnamaldehyde ⁄ ferricya-nide activity was determined, and related to the corresponding enzyme activity before the heat treatment step.
Fig 5 EPR spectra of YagTSR X-Band EPR spectra of reduced
YagTSR Experimental spectra are shown as solid black lines and
corresponding simulations as dotted black lines (A) Sodium
dithio-nite reduced YagTSR at T = 60 K with simulation of FeSI (B)
Sodium dithionite reduced YagTSR at T = 20 K with simulation of
FeSII (upper trace) and simulation of complete spectrum (lower
trace) (C) Treated with benzaldehyde (substrate) at T = 20 K The
obtained spectrum is identical to the spectrum shown in (B)
Exper-imental conditions: 1 mW microwave power (A), 0.25 mW
micro-wave power (B, C); 1 mT modulation amplitude, 12.5 kHz
modulation frequency, 9.56 GHz microwave frequency.
Trang 8with mutations in the yagTSRQ operon was not affected in comparison to the corresponding wild-type strain Cell growth was observed after a lag phase of approximately 4 h, and the stationary phase was initi-ated after approximately 15 h The addition of 800 lm cinnamaldehyde to the 0.1· LB medium (pH 4.0) led
to impaired cell growth of the wild-type, with a lag phase of 7 h The maximal attenuance obtained in the stationary phase reached only 35% of the value in the absence of cinnamaldehyde In comparison, mutations
in the yagR, yagT or yagQ genes resulted in complete impairment of cell growth in the presence of 800 lm cinnamaldehyde (Fig 6B) The phenotype is directly linked to the low pH in the medium, as at higher pH values of 6–7, no effect of the gene disruptions on cell growth in comparison to the wild-type strain was observed (data not shown) In addition, the growth of
E coli was also inhibited by the addition of 10 mm vanillin or 5 mm benzaldehyde to the 0.1· LB medium at pH 4.0 (data not shown)
Discussion
In this report, the molybdenum-containing iron–sulfur flavoprotein YagTSR from E coli was purified and characterized It was shown to be an aldehyde oxidore-ductase that oxidizes aldehydes to their respective acids The 135 kDa enzyme is a noncovalent hetero-trimer with a large (78.1 kDa) Moco-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2· [2Fe2S]-containing YagT subunit The YagT protein contains a 49 amino acid Tat leader peptide that allows the export of the active heterotrimer to the periplasm (data not shown) Tat substrates are matured in the cytoplasm prior to their translocation Here, we wanted to distinguish the translocation event from the Moco insertion into the YagTSR trimer Thus, we expressed the YagT protein without the Tat leader, resulting in a cytoplasmic and active YagTSR protein complex YagTSR purified from the cytoplasm contained 0.58 atoms of
moly-Table 4 g-Tensor principal values as obtained by simulation of experimental spectra g av = (g x + g y + g z ) ⁄ 3.
g-Values
Linewidth (mT)
a
The numbers in parentheses denote the g-strain used for simulation.bR capsulatus XDH, values taken from [36].
Fig 6 Growth curves of E coli BW25113, JW0278 (yagR)),
JW0280 (yagT)) and JW0277 (yagQ)) in the absence and presence
of 800 l M cinnamaldehyde E coli strains BW25113 (filled squares),
JW0278 (yagR), open squares), JW0280 (yagT), filled circles) and
JW0277 (yagQ), open circles) were inoculated at an attenuance at
600 nm of 0.1 in medium containing 1 gÆL)1 tryptone, 0.5 gÆL)1
yeast extract and 1 gÆL)1NaCl (0.1 · LB) at pH 4.0, and incubated
at 37 C and 150 r.p.m in the absence (A) and presence (B) of
800 l M cinnamaldehyde.
Trang 9bdenum, four atoms of iron, one molecule of FAD,
0.58 atoms of acid-labile sulfur, and 0.62 molecules of
CMP, showing that YagTSR binds the MCD form of
Moco This is the first enzyme identified in E coli that
binds the MCD So far, almost all characterized
E coli molybdoenzymes belong to the
dimethylsulfox-ide reductase family of molybdoenzymes, and have
been shown to bind bis-MGD The only enzyme that
does not belong to this class of molybdoenzymes is the
E coli YedY protein, which belongs to the sulfite
oxi-dase family and binds the Mo-MPT form of Moco [8]
Genetic investigations including the E coli xdhABC,
xdhDand yagTSRQ operons were performed by
Koz-min and Schaaper [39], who characterized the
resis-tance to N-hydroxylated base analogs in E coli They
classified the E coli xdhABC, xdhD and yagTSRQ
operons as putative family members of the XO family
However, without investigating the cofactor present in
these proteins, we believe that the authors erroneously
concluded that the enzymes bind the Mo-MPT form of
Moco [39] It is likely that the gene products of the
xdhABC and xdhD operons also bind the MCD form
of Moco, as an amino acid sequence alignment of the
Moco-binding subunits of E coli YagR, XdhA and
XdhD showed high amino acid identities to D gigas
MOP [12] and Oligotropha carboxidovorans CoxL [40],
two structurally characterized subunits that bind MCD
(Fig S1) In particular, the amino acids involved in
CMP binding are highly conserved in the three E coli
proteins (Fig S1)
We have additionally demonstrated that the activity
of YagTSR is independent of MobA, as an active
enzyme was purified from the mobAB-deficient E coli
TP1000 strain Thus, we conclude that MCD
biosyn-thesis in E coli requires a so far unidentified gene
product that catalyzes the attachment of CMP to
MPT Our investigations further showed that the
yagQ gene product is required for the production of
an active, Moco-containing YagTSR trimer When
YagTSR was expressed in the absence of YagQ, the
protein was inactive and devoid of Moco, and the
YagR subunit was shown to be unstable and was
rap-idly degraded during expression⁄ purification
There-fore, YagQ has a stabilizing effect on YagR and is
required for the insertion of Moco into the subunit
These characteristics are similar to those of the
R capsulatus XdhC protein, which is essential for the
insertion of Moco into XDH XdhC was reported to
be directly involved in the insertion of the sulfido
ligand of Moco while bound to XdhC, and,
further-more, to transfer the sulfurated cofactor to Moco-free
apo-XDH by direct interaction with the XdhB subunit
[33,41] XdhC seems to perform a ‘quality’ control of
Moco, as only sulfurated Moco is inserted into XDH
in R capsulatus We predict that YagQ performs a similar role for YagTSR in E coli, as YagTSR was shown to bind the sulfurated form of MCD However,
it needs to be clarified whether Moco bound to YagQ
is sulfurated before or after the attachment of the CMP moiety
The EPR spectra of YagTSR were found to be very similar to those of R capsulatus XDH, showing an almost axial EPR signal for FeSI and a broader, strongly rhombic signal for FeSII [36] Only subtle dif-ferences in the g-values and linewidths, in particular for the FeSI center (more rhombic g-tensor in YagTSR), are observed The values for gavare similar Neverthe-less, the overall close similarity of the EPR parameters indicated the presence of the same ligands and similar geometries of the two redox centers in YagTSR and
R capsulatus XDH The flavin semiquinone was not visible at pH 7.5; thus, it is not stabilized in YagTSR under physiological pH conditions This is similar to the situation with XOs, where the semiquinone is also not formed during reductive titrations, whereas in XDHs the flavin semiquinone is stabilized in the absence of NAD+[42,43] Here, the binding of NAD+ destabilizes the flavin semiquinone by increasing the redox potential of FADH⁄ FADH2 [43] As YagTSR does not use NAD+as electron acceptor, the stabiliza-tion of the flavin semiquinone seems to be less favor-able, which in this respect makes YagTSR more similar
to XOs Production of superoxide by YagTSR was also not observed, consistent with the observation that YagTSR does not use molecular oxygen as electron acceptor Thus, the physiological electron acceptor of YagTSR seems to react with fully reduced FAD Non-physiological electron acceptors such as 2,6-dichlor-ophenol-indophenol or ferricyanide were used by YagTSR However, as spinach ferredoxin was also used
as an electron acceptor, an E coli ferredoxin seems to
be a possible electron acceptor for this enzyme
Analysis of the substrate specificities of YagTSR showed a broad substrate spectrum with a preference for aromatic aldehydes, whereas purines were not oxi-dized by YagTSR The protein was shown to be stable
at low pH values between 4 and 5 Investigations of the phenotypes of single mutations in yagT, yagR and yagQ showed that YagTSR is essential under low-pH conditions in 0.1 · LB medium in the presence of the aromatic aldehydes cinnamaldehyde, vanillin, or benz-aldehyde Aromatic aldehydes occur ubiquitously in nature as flavoring and coloring molecules of plants, and during acidic and enzymatic degradation of lignin [44–46] Vanillin and cinnamaldehyde were shown to have antimicrobial activity at high doses [47–49]; for
Trang 10example, cinnamaldehyde is inhibitory to E coli
strains at concentrations of 250–500 lgÆmL)1 [47] It
was reported previously that cinnamaldehyde at a
con-centration of 500 lgÆmL)1 damaged the surface
struc-ture of E coli and thus resulted in impaired growth
[47] In addition, lower pH values (3.5–4.0) increased
the sensitivity to aromatic aldehydes of E coli [48]
Several potential mechanisms for the toxicity of
alde-hydes have been investigated, including damage from
chemical reactivity, direct inhibition of glycolysis and
fermentation, and plasma membrane damage
Alde-hydes are chemically reactive, and can form products
with many classes of biological molecules [50],
includ-ing nucleic acids, proteins, and lipids Our results show
conclusively that the periplasmic aldehyde
oxidoreduc-tase YagTSR is involved in the detoxification of
aro-matic aldehydes to their less toxic acids in the
periplasm of E coli, and cell damage under extreme
growth conditions is thus avoided
Experimental procedures
Bacterial strains, plasmids, media, and growth
conditions
Escherichia coliTP1000 (DmobAB) [35] was used for
homol-ogous expression of E coli yagTSRQ and yagTSR Cells
were grown aerobically in LB medium at 22C in the
pres-ence of 150 lgÆmL)1 ampicillin Sodium molybdate was
added at a concentration of 1 mm E coli strains BW25113,
JW0278 (yagR)), JW0280 (yagT)) and JW0277 (yagQ))
[51] were used for growth experiments
Cloning, expression and purification of E coli
YagTSRQ
DNA fragments containing the coding regions for E coli
yagTSRQ were amplified by PCR from total DNA
obtained from E coli K-12 The flanking restriction sites
NdeI and SacI were introduced by the PCR primers, and
the amplified yagTSRQ operon was cloned without the
N-terminal Tat leader sequence into the NdeI–SacI sites of
the expression vector pTrcHis [52] The resulting plasmid
pMN100 expresses the yagTSRQ operon as an N-terminal
His6-tag fusion to YagT for affinity purification from the
cytoplasm A plasmid expressing only the yagTSR operon
in pTrcHis, named pMN111, was obtained by deletion of
the yagQ gene in plasmid pMN100
For production of His6-tagged YagTSR, E coli TP1000
cells were transformed with plasmids pMN100 and
pMN111, respectively One liter of LB supplemented with
1 mm sodium molybdate and 10 lm isopropyl
thio-b-d-galactoside was inoculated with 2 mL of an overnight
culture and incubated for 24 h at 22C and 100 r.p.m The
cells were harvested by centrifugation at 9600 g for 5 min The cell pellet was resuspended in phosphate buffer (50 mm NaH2PO4, 300 mm NaCl, pH 8.0) Complete cell lysis was achieved by three passages through a TS Series Benchtop cell disruptor (Constant Systems, Daventry, UK) at 1350 bar in the presence of DNaseI (1 lgÆmL)1) The cleared lysate was applied to 0.5 mL of Ni2+–nitrilotriacetic acid (Qiagen, Hilden, Germany) per liter of culture The column was washed with 2· 20 column volumes of phosphate buf-fer containing 10 and 20 mm imidazole each Protein was eluted with phosphate buffer containing 250 mm imidazole, and the buffer was changed to 50 mm Tris and 1 mm EDTA (pH 7.5) by either dialysis or PD10 gel filtration chromatography (GE Healthcare, Munich, Germany) For further purification, the YagTSR was applied to a Q-Sepha-rose column (GE Healthcare) and eluted with a linear gra-dient of 0–1 m NaCl in 50 mm Tris and 1 mm EDTA (pH 7.5) Size exclusion chromatography using 0.3 mg of YagTSR was performed using a Superdex 200 column (GE Healthcare) with a bed volume of 24 mL equilibrated in
50 mm Tris and 200 mm NaCl (pH 7.5) The size of YagTSR was determined by using a gel filtration standard (BioRad, Hercules, CA, USA) To determine the purity of YagTSR, densitometric analysis of Coomassie Brilliant Blue-stained SDS⁄ PAGE gels was performed using quantityone 4.6 software (BioRad) The YagTSR concen-tration of the purified enzyme was determined from the absorbance at 445 nm, using an extinction coefficient of
23 686 m)1Æcm)1 for the native enzyme The extinction coefficient was determined on the basis of FAD content after trichloroacetic acid precipitation [53]
Metal analysis Metal analysis was performed using PerkinElmer Optima 2100DV ICP-OES (Fremont, CA, USA) Protein samples were wet-ashed overnight in a 1 : 1 mixture with 65% nitric acid (Suprapur; Merck, Darmstadt, Germany) at 100C Samples were diluted with 4 mL of H2O prior to their injection onto the ICP-OES apparatus As reference, the multielement standard solution XVI (Merck) was used
Moco⁄ MPT analysis
To determine the MCD content of YagTSR, the samples were incubated overnight at room temperature in the pres-ence of acidic iodine to convert MCD to Form A CMP Form A was separated from Form A using a 400 lL Q-Sepharose ion exchange column (GE Healthcare), which was equilibrated in H2O The oxidized samples were loaded, and Form A was eluted with 0.8 mL of 10 mm ace-tic acid and analyzed as described previously [41] Form A was eluted with 0.6 mL of 50 mm HCl and directly applied
to a C18 RP-HPLC column (4.6· 250 mm ODS Hypersil, particle size 5 lm; Thermo Scientific, Karlsruhe, Germany),