All F420-dependent enzymes analysed to date in this respect have been shown to be Si-face stereospecific at C5 of F420 [6].. All the coenzyme F420-dependent enzymes investigated to date h
Trang 1oxidase from methanogenic Archaea as determined by
mass spectrometry
Henning Seedorf1, Jo¨rg Kahnt1, Antonio J Pierik2and Rudolf K Thauer1
1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2 Fachbereich Biologie, Philipps-Universita¨t, Marburg, Germany
Methanogenic Archaea fluoresce greenish yellow when
irradiated with UVA light The fluorescence is due to
coenzyme F420, a
7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative (Fig 1) The coenzyme, which is
generally present in 1 mm intracellular concentrations
[1], functions as a redox mediator in methanogenesis,
in NADP+ reduction and in glucose-6-phosphate
dehydrogenation [2] With respect to its redox
proper-ties, F420 is much more similar to pyridine nucleotides
than to flavins [3] Both F420 and NAD(P) transfer
hydride anions and not single electrons In the reduced
form, both F420 and NAD(P) have a prochiral centre,
F420 at C5 and NAD(P) at C4 They differ
function-ally, mainly in that the redox potential of the
F420⁄ F420H2 pair (E¢ ¼)360 mV) is 40 mV more
negative than that of the NAD(P)+⁄ NAD(P)H pair (E¢ ¼)320 mV) [5]
All F420-dependent enzymes analysed to date in this respect have been shown to be Si-face stereospecific at C5 of F420 [6] This is surprising because NAD(P)-dependent enzymes can be Si-face or Re-face specific [7] and some flavoenzymes, whose apoproteins catalyse the reduction of synthetic 8-hydroxy-5-deaza-FAD, have been shown to be Si-face stereospecific with respect to C5 of the synthetic deazaflavin and others
to be Re-face stereospecific [7,8] In case of the pyridine-nucleotide-dependent enzymes the redox potential (E) of the electron acceptor reduced by NAD(P)H is thought to be an important factor deter-mining the stereospecificity of these enzymes [9,10]
Keywords
coenzyme F 420 ; 5-deazaflavins;
F420H2oxidase; methanogenic Archaea;
stereospecificity
Correspondence
R K Thauer, Max Planck-Institute for
Terrestrial Microbiology,
Karl-von-Frisch-Strasse, D-35043 Marburg, Germany
Fax: +49 642 117 8109
Tel: +49 642 117 8101
E-mail: thauer@staff.uni-marburg.de
(Received 4 July 2005, revised 17 August
2005, accepted 23 August 2005)
doi:10.1111/j.1742-4658.2005.04931.x
Coenzyme F420 is a 5-deazaflavin Upon reduction, 1,5 dihydro-coenzyme
F420 is formed with a prochiral centre at C5 All the coenzyme
F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420 -dependent enzymes being unrelated phylogenetically In this study, we report that the recently discovered F420H2 oxidase from methanogenic Archaea is also Si-face stereospecific The enzyme was found to catalyse the oxidation of (5S)-[5-2H1]F420H2 with O2 to [5-1H]F420 rather than to [5-2H]F420 as determined by MALDI-TOF MS (5S)-[5-2H1]F420H2 was generated by stereospecific enzymatic reduction of F420 with (14a-2H2 )-[14a-2H2] methylenetetrahydromethanopterin
Abbreviations
Adf, F420-specific alcohol dehydrogenase; F420, coenzyme F420; Fgd, F420-dependent glucose-6-phosphate dehydrogenase; Fno, F420H2: NADP + oxidoreductase; Fpo, F 420 H 2 dehydrogenase complex; FprA, F 420 H 2 oxidase; Frd, F 420 -dependent formate dehydrogenase; Frh,
F 420 -reducing hydrogenase; H 4 MPT, tetrahydromethanopterin; Mer, F 420 -dependent methylenetetrahydromethanopterin reductase;
methylene-H4MPT, methylenetetrahydromethanopterin; Mtd, F420-dependent methylenetetrahydromethanopterin dehydrogenase; TFA, trifluoroacetic acid.
Trang 2Redox potentials (E) of the electron acceptor more
neg-ative than )200 mV generally promote Si-face
stereo-specificity and redox potentials more positive than
)200 mV promote Re-face stereospecificity at C4
of NAD(P) Thus the NADP-dependent
glucose-6-phos-phate dehydrogenase is Si-face specific (E ¼
)330 mV) and the NAD-dependent malate
dehydro-genase is Re-face specific (E ¼)170 mV) Most
eth-anol dehydrogenases are Re-face specific (E ¼
200 mV), but the enzyme from Drosophila melanogaster
is Si-face specific For recent literature on the subject see
Berk et al [11]
The following eight F420-dependent enzymes have
been analysed and shown to be Si-face specific: F420
-reducing hydrogenase (Frh) [12,13]; F420-dependent
formate dehydrogenase (Frd) [14]; F420-specific alcohol
dehydrogenase (Adf) [6]; F420-dependent
methylene-tetrahydromethanopterin dehydrogenase (Mtd) [15,16];
F420-dependent methylenetetrahydromethanopterin
reductase (Mer) [15]; F420H2 dehydrogenase complex
(Fpo) [15]; F420H2: NADP+ oxidoreductase (Fno)
[17,18]; and F420-dependent glucose-6-phosphate
de-hydrogenase (Fgd) [6] Adf, Mer and Fgd form a
fam-ily, as do the F420-binding subunits FpoF, FrhB and
FrdB The two families, Mtd and Fno are not
phylo-genetically related The eight enzymes catalyse redox
reactions with electron acceptors ranging in redox
potential (E¢) from )414 mV (2H+
⁄ H2) to )165 mV (methanophenazine ox⁄ red) [19] The Si-face
stereospe-cificity of F420-dependent enzymes thus appears to be
independent of their phylogenetic origin and of the
thermodynamics of the reactions catalysed by them
Recently a novel F420-dependent enzyme, F420H2
oxidase (FprA), was discovered in methanogenic
Arch-aea [20] FprA catalyses the oxidation of 2 F420H2with
O2 to 2 F420and 2 H2O The 45 kDa protein contains
1 FMN per mol and harbours a binuclear iron centre
indicated by the sequence motif H-X-E-X-D-X63
-H-X18-D-X62-H FprA is not phylogenetically related
to any of the other F420-dependent enzymes and cata-lyses a reaction with a redox potential difference
of +1.27 V (F420H2 oxidation with O2) We therefore investigated the stereospecificity of this enzyme and found it to be Si-face specific at C5 of F420 The method used was based, in principle, on the technique for determining the hydride transfer stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreduc-tases by MS [21]
Results
The following findings are important for the under-standing of the results shown in Fig 2: (a) [5-1H]F420 and [5-2H]F420 can be identified and the relative amounts present in a mixture quantitated using MALDI-TOF-MS; (b) F420H2 auto-oxidizes nonstereo-specifically to F420 in the matrix used for MALDI-TOF-MS; (c) F420 is stereospecifically reduced to
F420H2 with methylenetetrahydromethanopterin (methylene-H4MPT) in the presence of F420-dependent methylene-H4MPT dehydrogenase (Mtd), which has been shown to be Si-face specific at C5 of F420[15,16]; and (d) F420 is chemically reduced to F420H2 with NaBH4in a nonstereospecific reaction
In Fig 2A three MALDI-TOF mass spectra of a control experiment are shown: the spectrum of [5-1H]F420 (Fig 2Aa); the spectrum of [5-1H2]F420H2 generated from [5-1H]F420 by Mtd-catalysed reduction with [14a-1H2]methylene-H4MPT (Fig 2Ab); and the spectrum of [5-1H]F420 generated from [5-1H2]F420H2
by FprA catalysed oxidation (Fig 2Ac) As seen from the normalized 1 Da separated stick spectra (insets, black) all three mass spectra are almost identical to the stick spectrum (insets, white) calculated for [5-1H]F420 from its elemental composition considering the isotope composition of the elements: 98.9% 12C, 1.1% 13C; 99.63% 14N, 0.37% 15N; 99.99% 1H, 0.01% 2H; and 99.76% 16O, 0.24%17O and18O
The experiment shown in Fig 2B differs from that
in Fig 2A only in that in the first step F420 was enzy-matically reduced with [14a-2H2] methylene-H4MPT yielding (5S)-[5-2H1]F420H2 FprA catalysed oxidation
of (5S)-[5-2H1]F420H2 yielded only[5–1H]F420 as indica-ted by the mass spectrum (Fig 2Bc), which was identi-cal to that identi-calculated for [5-1H]F420 (Fig 2Ba) This result can only be explained if FprA is Si-face specific with respect to C5 of F420 In contrast, auto-oxidation
of (5S)-[5-2H1]F420H2 yielded a 1 : 2 mixture of [5-1H]F420 and [5-2H]F420, as indicated by the relative intensities of the 772 and 773 Da mass peaks
Fig 1 Structure of reduced coenzyme F 420 (F 420 H 2 ) F 420 ¼
N-(N-L -lactyl- L -glutamyl)- L -glutamic acid phosphodiester of
7,8-didemethyl-8-hydroxy-5-deazariboflavin.
Trang 3(Fig 2Bb, stick spectrum, black) For comparison the
relative intensities calculated for a 1 : 1 mixture are
given (Fig 2Bb, stick spectrum, white) The [5-1H]F420
to [5-2H]F420 ratio of 1 : 2 can be explained assuming
a deuterium isotope effect of 2 for the
auto-oxida-tion reacauto-oxida-tion
As a control, F420 was reduced with NaB2H4
(NaBD4) yielding a mixture of (5S)-[5-2H1]F420H2 and
(5R)-[5-2H1]F420H2 The FprA-catalysed oxidation of
the mixture yielded a 1 : 1 mixture of [5-1H]F420 and
[5-2H]F420as revealed by the relative intensities of the
772 and 773 Da mass peaks (Fig 2Cc) The results are
consistent with FprA catalyzing the oxidation of
(5S)-[5-2H1]F420H2to [5-1H]F420 and the oxidation of
(5R)-[5-2H1]F420H2 to [5-2H]F420 as to be expected for a
Si-face-specific enzyme The finding that the 775 Da
mass peak in Fig 2Cb was much lower than in
Fig 2Bb is probably due to the fact that reduction
of F420 with NaBD4 (Fig 2C) was not complete
and therefore after auto-oxidation the F420 analysed
contained less2H
Discussion
In the Introduction it was pointed out that all F420
-dependent enzymes investigated have been shown to be
Si-face specific at C5 of F420, despite four of these
enzymes being unrelated phylogenetically The finding
that F420H2oxidase (FprA) is also Si-face specific brings
to five the number of Si-face-specific F420-dependent
enzymes that are not related phylogenetically There is only a 6.25% probability that this is by chance
To date, the crystal structures of four F420-dependent enzymes have been resolved: F420H2:NADP oxido-reductase, with and without F420 bound [22];
F420-dependent alcohol dehydrogenase with F420bound [23]; Mer, with and without F420 bound [24,25]; and Mtd without F420bound [26] A common F420-binding
Fig 2 MALDI-TOF-MS analysis of F420and F420H2for the
deter-mination of the stereospecificity of F420H2 oxidase (FprA) The
insets show normalized 1 Da separated stick spectra obtained from
the measured data (black) aligned to simulated spectra (white) For
better visibility in the structures deuterium is abbreviated by D
rather than by 2 H and hydrogen by H rather than 1 H Mtd,
Si-face-specific F420-dependent methylenetetrahydromethanopterin
de-hydrogenase (A) Experiment with nonlabelled substrates showing
that the mass spectrum of [5- 1 H 2 ]F 420 H 2 (b), owing to
auto-oxida-tion of F 420 H 2 , is identical to that of [5-1H]F 420 (a, c) The simulated
stick spectra (white) are for [5- 1 H]F420 (B) Experiment with
specif-ically 2 H-labelled substrates showing that the mass spectrum of
F 420 formed from (5S)-[5-2H 1 ]F 420 H 2 by FprA-catalysed oxidation (c)
is identical to the spectrum of [5- 1 H]F420 (a) The simulated stick
spectrum (b, white) of (5S)-[5- 2 H1]F420H2 is for a 1 : 1 mixture of
[5-1H]F 420 and [5-2H]F 420 The other two (a, c) are for [5-1H]F 420 (C)
Experiment with NaB 2 H4-reduced [5- 1 H]F420showing that the mass
spectrum of F420formed from reduced F420by FprA-catalysed
oxi-dation corresponds to that of a mixture of [5- 1 H]F 420 and [5- 2 H]F 420
(c) The simulated stick spectrum of reduced F420(b, white) and
that of the FprA oxidation product (c, white) are for a 1 : 1 mixture
of [5- 1 H]F 420 and [5- 2 H]F 420
Trang 4motif explaining the Si-face specificity of these enzymes
was not found It therefore has to be considered that
Si-face specificity may be an intrinsic property of F420
rather than of the F420-dependent enzymes An example
of the stereospecificity of a dehydrogenase being
dicta-ted by the structure of its substrate has recently been
published There are several phylogenetically unrelated
methylenetetrahydromethanopterin dehydrogenases
and methylenetetrahydrofolate dehydrogenases that are
all Re-face specific at the carbon of the methylene group
[27,28] It has been calculated that the transition state
conformation of methylenetetrahydromethanopterin
and methylenetetrahydrofolate for the dehydrogenation
from the Re-face is energetically favoured [28]
How-ever, in the case of F420, there are no centres of
asym-metry in the near neighbourhood of C5 that could
interact with the reactant or the product, or affect the
transition state(s) and by that induce an intrinsic
ener-getic difference in the reaction profiles involving the Si
versus the Re side of F420 The nearest asymmetry
cen-tres are in the N10 side chain It is therefore difficult
to envisage how the Si-face stereospecificity of F420
-dependent enzymes could be dictated by the structure
of F420
Experimental procedures
Isotopes, coenzymes and enzymes
Deuterium oxide (2H2O) and deuterated formaldehyde
(2H2CO) were from Euriso-Top (Saarbru¨cken, Germany)
and sodium borodeuteride (NaB2H4) was from Fluka
(Tauf-kirchen, Germany) Coenzyme F420 and
tetrahydrometha-nopterin (H4MPT) were purified from Methanothermobacter
marburgensis(DSMZ 2133) [29] [14a-1H2]methylene-H4MPT
was prepared by spontaneous reaction of H4MPT and
1
H2CO and [14a-2H2]methylene-H4MPT by spontaneous
reaction of H4MPT and2H2CO [30] FprA from M
marbur-gensis [20] and Mtd from Methanopyrus kandleri [26] were
produced heterologously in Escherichia coli and purified to
specific activities of 100 and 4000 UÆmg)1, respectively
(1 U¼ 1 lmolÆmin)1) Protein was determined with the
Rot-Nanoquant-Microassay from Roth (Karlsruhe, Germany)
using bovine serum albumin as standard
Assay to determine the stereospecificity of F420H2
oxidase
The assay is described in Fig 2B The 1.2 mL assay
mix-ture at 30C contained 60 lm H4MPT, 140 lm2H2CO and
55 lm F420 in oxic 120 mm potassium phosphate pH 6
Reduction of F420to (5S)-[2H1]F420H2with [14a-2H2
]methy-lene-H4MPT (spontaneously generated from H4MPT and
2H2CO) was started by the addition of 120 U Mtd (Si-face specific) and was completed after 5 min Subsequently,
60 U FprA were added, which catalysed the oxidation of
F420H2 with O2 as the electron acceptor Samples of the assay were taken before and 5 min after the addition of Mtd and 5 min after the addition of FprA and analysed by MALDI-TOF-MS
In the control experiment described in Fig 2A, the 1.2 mL assay mixture contained 140 lm 1H2CO instead of
140 lm2H2CO
In the control experiment described in Fig 2C, the 1.2 mL assay did not contain H4MPT, H2CO or Mtd Instead, F420 was reduced with NaB2H4 to a mixture of (5S)-[5-2H1]F420H2 and (5R)-[5-2H1]F420H2 This step was carried out under anaerobic conditions
Analysis of F420and F420H2by MALDI-TOF-MS
Samples (25 lL) of the 1.2 mL assay mixtures were applied
to a small ZipTips (Millipore Corp, Bedford, MA, USA) column previously equilibrated with 0.1% (v⁄ v) trifluoro-acetic acid (TFA) The column was then washed with 0.1% (v⁄ v) TFA to remove salts and was then eluted with 84% (v⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA The eluate was dried by vacuum centrifugation and the dried pellet dissolved in
10 lL 0.1% (v⁄ v) TFA and subsequently supplemented with 10 lL of a saturated solution of a-cyano-4-hydroxy-cinnamic acid in 70% (v⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA Aliquots were air dried and analysed by MALDI-TOF-MS The mass spectra were collected in the reflector negative-ion mode For each spectrum, at least 150 single shots were summed The spectra were determined with a Voyager DE
RP from PE Biosystems
The natural isotopic distribution in F420 was calculated
by the isotope pattern calculator provided by the University
of Sheffield at the ChemPuter site (http://www.shef.ac.uk/ chemistry/chemputer/) All calculations of simulated data were carried out in excel 2000 and transformed into stick spectra separated by 1 Da [31]
Acknowledgements
This work was supported by the Max Planck Society and by the Fonds der Chemischen Industrie Henning Seedorf thanks the Deutsche Forschungsgemeinschaft for a graduate fellowship We are indebted to Christ-oph Hagemeier for providing purified F420-dependent Mtd from Methanopyrus kandleri
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