6 Characterisation of resorcinol (1,3-dihydroxybenzene) hydroxylase and detection of hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ)
6.3.6 Analytical methods
Nitrite was determined chemically with commercial available analytical tests strips (Merck, Germany).
6.4 Results
6.4.1 Molybdenum dependency in growth experiments
A. anaerobius was cultivated in molybdate-free medium with resorcinol as carbon and energy source with either nitrate or nitrous dioxide as electron acceptor. Transfers of A. anaerobius (5 %) in fresh molybdate-free medium were done in the late exponential growth phase. Although the medium lacked molybdenum all cultures grew as well as in complete medium. The growth rate was 0.03-0.06 h-1 and the cultures reached a final optical density at 578 nm (OD578) at 0.4 to 0.6. Cells in each transfer were uniformly rod-shaped, as described before (Springer et al., 1998). All bacteria were motile and occurred mostly in pairs. Addition of the molybdate antagonist tungstate (0.02 mM) (Blaschke et al., 1991) inhibited growth with resorcinol completely (Fig. 1, a and b). In cultures with nitrate, no nitrite formation was detected. The few bacterial cells visible were heavily deformed and tended to form aggregates.
These cells were immotile. Addition of molybdate (0.02 mM) caused an increase in OD578 and a formation of nitrite (Fig.1, a and b), and the cells returned to normal.
The same experiment was performed with acetate as growth substrate and nitrate or nitrous dioxide as electron acceptor. The increase of OD578 in growth experiments with nitrate as electron acceptor was slightly retarded after addition of tungstate (Fig. 1 c). Addition of molybdate had no influence on the increase of OD578. The increase of OD578 in growth experiments with nitrous oxide as electron acceptor was not affected by addition of tungstate or molybdate (Fig. 1 d).
Fig. 1 Increase of optical density at 578 nm of A. anaerobius without tungstate (♦) with tungstate (? ) and with tungstate supplemented with molybdate after 1 day of incubation (¦) grown with resorcinol (a and b) or acetate (c and d) as carbon and energy source and nitrate (a and c) or nitrous dioxide (b and d) as electron-acceptor.
Further growth experiments were performed with A. anaerobius with either resorcinol or acetate as carbon and energy source and nitrate as electron acceptor, supplemented with inhibitors for molybdenum-containing enzymes.
Arsenite or selenite (0.02 mM) (Lehmann et al., 1994) had no effect on growth.
In all cultures, the increase of OD578 and the cell morphology were identical to the control without addition of any inhibitor.
6.4.2 Resorcinol hydroxylase in the presence of inhibitors
The specific activity of membrane-bound resorcinol hydroxylase (RH) was measured in the presence of inhibitors for molybdenum-containing enzymes or in the presence of the molybdate antagonist tungstate (Tab. 1). Arsenite and
(a)
selenite decreased the specific activity of RH, whereas cyanide and tungstate had a positive effect.
Tab. 1 The specific activity of the resorcinol hydroxylase in membrane fraction of A. anaerobius grown on resorcinol with inhibitors for molybdenum containing enzymes and with molybdenum antagonist tungstate.
Substance Concentration
[mM]
RH activity [U/mg protein]
Activity [%]
Reference - 0,30 100
Arsenite 1 0,01 3
Selenite 1 0,12 39
KCN 1 0,53 150
Tungstate 0,02 0,42 135
6.4.3 Detection of HHQ dehydrogenase
Protein expression patterns of the membrane fractions were compared in a SDS-PAGE after growth with either succinate or resorcinol (Fig. 2, line 1, 2).
Eight prominent protein bands were observed in the membrane fraction of resorcinol-grown cells. These proteins had molecular masses of approximately 42, 50, 58, 70, 80, 90, 105 and 120 kDa.
Fig. 2 SDS-PAGE of membrane fractions of A. anaerobius grown on succinate (1) or resorcinol (2), T. aromatica AR-1 R+ grown on succinate (3) or resorcinol (4) and T. aromatica K 172 R+ grown on succinate (5) or resorcinol (6). MM represents the molecular weight marker in kDa. The protein with the mass of 50 kDa has been marked.
The protein with the mass of 50 kDa was subjected to direct N-terminal and internal amino acid sequencing. The N-terminus consisted of 13 amino acids and the internal sequences of 7, 8, and 13 amino acids (Tab. 2). Other peptides gave no reliable results.
Tab. 2 N-terminal and internal amino acid sequences of protein 50 kDa isolated form a SDS-PAGE of membrane fraction from A. anaerobius grown on resorcinol. Amino acids in brackets indicate tentative assignment.
Position Amino acid sequence Length [amino acids]
N-terminus M-H-K-G-L-F-(G)-P-I-E-L-R-(G) 13
internal F-G-V-A-A-Q-G-I/L-Q-I/L-A-H-A 13
internal E-E-Q-D-D-H-V-E 8
internal F-A-I/L-E-T-F-E 7
The oligopeptides of the protein of 50 kDa molecular mass aligned only poorly with deposited protein sequences accessible on the NCBI server. However, these oligopeptides aligned to one translated open reading frame (ORF 21) of the recently sequenced genome fragment R+ of A. anaerobius (Tab. 3) (Hellstern et al., unpublished (a)).
Tab. 3 Consensus sequence of protein 50 kDa isolated from a SDS-PAGE of membrane fraction from A. anaerobius grown on resorcinol, translated ORF 21 from sequence R+ of A. anaerobius and NADH: flavin oxidoreductase of Thiobacillus denitrificans ATCC15159. The identical amino acids are highlighted. The location of the amino acids of each consensus sequence is indicated.
Consensus sequence N-terminal internal internal internal
Protein 50 kDa 1 MHKGLFGPIELRG FGVA_AGGIQLAHA EEQDDHVE FALETFE Translated ORF 21 1 MHNKLFSPIELRG 93 FGVA_AQGIQLAHA 155 QIQDDHVE 210 FALETFE NADH:flavin
oxidoreductase
1 M_STLFSPFRLGT 92 YAPPLALGIQLAHA 155 RVRGEFVT 210 FPLEVFD
Translated ORF 21 revealed 51 % identity to an NADH: flavin oxidoreductase of Thiobacillus denitrificans (Fig. 3). It was predicted to be involved in the HHQ dehydrogenation (Hellstern et al., unpublished (a)).
Prot.: 4 LFSPFRLGTRVLKNRIVVSPMCQYSAEDGCATDWHMIXXXXXXXXXXXXXFVEATAVSPE 63 LFSP L LKNR+VVSPMCQY +E+G A DWH+ EAT V+
ORF21: 5 LFSPIELRGLTLKNRLVVSPMCQYISENGSANDWHLYHLGNFSIGGFGLVMTEATNVNTV 64
Prot.: 64 GRIAASDLGLWSDDNEAALRRVVDAIRRYAPPLALGIQLAHAGRKASTDVPWAGGHFILP 123 G+I L +D+NEAAL+RV+D +++ A GIQLAHAGRK+ST P GGH + ORF21: 65 GKITHKCATLCTDENEAALKRVIDFNKKFG-VAAQGIQLAHAGRKSSTHPPALGGHPLEA 123
Prot.: 124 EQGGWQTLAPSALAYAPGDAAPRALDTAGLERVRGEFVTAAQRAERLGFDVIELHAAHGY 183 + G W+T+APSA+ YA G PRAL A +++++ + V + +RA R+G+D+IE+HA HGY ORF21: 124 DDGAWETVAPSAIPYAAGWHVPRALSKAEIQQIQDDHVESVKRALRIGYDLIEMHAGHGY 183
Prot.: 184 LLHEFLSPLSNVRDDRYGGSLENRMRFPLEVFDAMRAALHADVPLGVRISATDWVEGGWD 243 L H+FLSPL+N R D YGGSL+NRMRF LE F+AMR D P+G R+SA+DWVEGGW ORF21: 184 LTHQFLSPLANQRTDEYGGSLQNRMRFALETFEAMREVWPKDKPMGARVSASDWVEGGWT 243
Prot.: 244 IAQSLAFAEALRRRGCAFIDVSSGGLSPAQQIPVAPGYQLPFAAQIRGETGMPTMAVGLI 303 + +++AFA LR+ GC +IDVSSGGL PAQ +P+AP Y A +IR E + M VGLI ORF21: 244 VDETVAFARELRKLGCDYIDVSSGGLHPAQAVPLAPAYHAEIAQRIRNEADIKVMVVGLI 303
Prot.: 304 TDPHQAENIVAGGHADLVALARGMLYDPRWPWHAAAELGARADAPKQYWAAPPHGHGTLF 363 DPH A IV G AD V + RG ++DPR+PWHAA GA A P + P LF ORF21: 304 ADPHVATKIVESGQADFVCMGRGAMWDPRFPWHAAETFGAEAQYPARSTPCLPKLRPQLF 363
Prot.: 364 KRDKD 368 ++
ORF21: 364 PNHQE 368
Fig. 3 Alignment of NADH:flavin oxidoreductase of Thiobacillus. denitrificans (Prot.) and ORF 21 of genome fragment R+ of A. anaerobius.
The protein with the mass of 50 kDa was also detected in membrane fractions of Thauera aromatica AR-1, a facultatively nitrate-reducing bacterium harbouring the HHQ-degradation pathway (Fig. 4, line 2).
Fig. 4 SDS-PAGE of membrane fractions of A. anaerobius grown on resorcinol (1), T. aromatica AR-1 grown on α -resorcylate (2) and T. aromatica AR-1 R+ grown on benzoate (3), α--resorcylate (4), resorcinol (5) and succinate (6).
It was not detected in membrane fractions of the facultatively nitrate-reducing bacterium T. aromatica K 172 (Fig. 5, line 1), which is not known to possess the HHQ-degradation pathway.
Fig. 5 SDS-PAGE of membrane fractions of T. aromatica K172 wt grown on benzoate (1), A. anaerobius grown on resorcinol (2) and T. aromatica K172 R+ grown on resorcinol (3).
However, the protein with 50 kDa molecular mass was detected in the transconjugante T. aromatica K 172 R+ enabled to use resorcinol by a cosmid containing the genome fragment R+ of A. anaerobius. This fragment contains genes involved in resorcinol degradation by A. anaerobius (Hellstern et al., unpublished (a)). The 50 kDa protein was present in membrane fractions of cells grown with succinate as well as in cells grown with resorcinol (Fig. 2, line 5, 6), but expression of this protein was up-regulated after growth with resorcinol.
The sequence R+ enabled also transconjugante T. aromatica AR-1 R+ to use resorcinol as energy and carbon source after heterologous expression. The 50 kDa protein of the transconjugante AR-1 R+ was present at higher amounts after growth with resorcinol or α-resorcylate compared to wildtype T. aromatica AR-1 grown on α-resorcylate (Fig. 4 line 2, 4, and 5), and compared to transconjugante K 172 R+ and A. anaerobius grown with resorcinol (Fig. 2 line 2, 4, 6).
6.4.4 Proteins involved in resorcinol degradation
Besides the 50 kDa protein further induced proteins were detected in SDS-PAGE of membrane fractions of A. anaerobius, T. aromatica AR-1 R+ and K 172 R+ grown with resorcinol. They were 42, 80 and 105 kDa in mass (Fig.
2). The protein with 42 kDa molecular mass was present only in membrane fractions of resorcinol-grown cells, whereas the proteins with the mass of 80 and 105 kDa were also present in T. aromatica AR-1 grown with α-resorcylate (Fig. 4, line 2).
6.5 Discussion
Resorcinol hydroxylase (RH) of A. anaerobius oxidizes resorcinol (1,3-dihydroxybenzene) to hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ) (Philipp and Schink, 1998). Molecular oxygen was not available under the conditions applied and the required oxygen should thus derive from water.
Hydroxylating enzymes incorporating an oxygen atom from water are known to be molybdenum-containing enzymes (Hille et al., 1999). The first attempt to prove the necessity of molybdenum during resorcinol degradation was to starve A. anaerobius for molybdenum in a medium lacking this trace element.
However, also dissimilatory nitrate reductase of denitrifiers requires a molybdenum atom in their catalytically active centre (Zumft, 1997). In order to exclude growth inhibition to the debit of the nitrate reductase, either nitrate or nitrous oxide was used as electron acceptor. No inhibition of growth was observed in neither one of these setups after several transfers. Obviously, molybdenum was still available, possibly provided by the used Milli-Q water.
The minimal molybdate concentration for supply of nitrogenases is 5 µM (Brill et al., 1974) and for nitrate reductases 0.001 µM (Pau et al., 1997). Therefore, A. anaerobius must have a very high affinity molybdenum uptake system completed by a molybdenum accumulation system, as this was described for Azobacter vinelandii, Escherichia coli, and Klebsiella pneumonia (Pau et al., 1997) in order to provide enough molybdenum for enzymes participating in resorcinol metabolism.
Tungstate was added to the growth medium so that the low but troublesome concentration of molybdenum was no longer of any relevance. Tungsten is known to be a molybdenum antagonist (Blaschke et al., 1991). The size, the shape as well as the chemical and physical properties of molybdate and tungstate ions are very similar (Johnson, 1988). At excessive supply with tungstate, the nitrate reductase of E. coli incorporates tungstate yielding an
inactive enzyme form (Amy and Rajagopalan, 1979). The inhibitory effect of tungstate on the increase of OD578 of A. anaerobius was observed only in cultures grown with resorcinol, independent of the used electron acceptor.
During growth with acetate plus nitrate a slight delay in OD578 was observed, which was probably due to insufficient molybdenum supply for the nitrate reductase (Zumft, 1997). The negligible effect of tungstate on the nitrate reductase but the inhibitory effect on growth with resorcinol could be explained by the fact that nitrate reductase plays a central role in growth under denitrifying conditions. The nitrate reductase is responsible for maintenance of the entire energy metabolism of the cells whereas RH had to sustain `only´ the oxidation of resorcinol. Therefore, RH did not obtain the highest priority in molybdenum supply at limitation.
Addition of arsenite or selenite, inhibitors for molybdenum-containing enzymes (Lehmann et al., 1994), had no negative impact on the growth behaviour of A. anaerobius with resorcinol as growth substrate. However, in cell-free extract experiments arsenite and selenite inhibited resorcinol hydroxylase tremendously. Both inhibitors can destabilize the molybdenum bound in the protein by forming a complex with the thiol groups of the enzyme (Voet and Voet, 1994). Thereby, the enzyme looses its tertiary structure and is unable to perform its catalytic reaction. However, cyanide, a prominent inhibitor for molybdenum-containing enzymes (Coughlan et al., 1980), did not inhibit resorcinol hydroxylase. This could be due to the aldehydes and keto acids arising during resorcinol degradation (Hellstern et al., unpublished (b)), which add to cyanide to form cyanohydrins (Organikum, 1970), thereby render cyanide harmless.
Comparison of protein patterns of membrane fractions of A. anaerobius grown with different substrates revealed eight protein bands which either were visible only in membrane fractions of cells grown with resorcinol, or the band intensity increased after growth with resorcinol. A 50 kDa protein belonged to the latter.
This protein was isolated and partially sequenced. The sequences of the oligopeptides aligned to a translated open reading frame (ORF) of the recently described genome fragment R+ of A. anaerobius. This genome fragment R+
contains the genes for resorcinol hydroxylase, HHQ dehydrogenase, and probably the genes for further degradation to acetate, malate, and succinate
(Hellstern et al., unpublished (a)). Membrane fractions of two transconjugantes Thauera aromatica AR-1 R+ and K 172 R+, containing the genome fragment R+ of A. anaerobius, contained the 50 kDa protein, too. It was constitutively expressed although its concentration was increased after growth with resorcinol. However, the transconjugante AR-1 R+ expressed this protein at higher concentration than wildtype T. aromatica AR-1, A. anaerobius, and the transconjugante K 172 R+. This is due to the fact that wild-type (wt) T. aromatica AR-1 degrades α-resorcylate over a hydroxylation followed by a decarboxylation to the central intermediate HHQ (Gallus and Schink, 1998).
The subsequent dehydrogenation of HHQ to HBQ is assumed to be catalysed by a similar HHQ dehydrogenase in T. aromatica AR-1 and A. anaerobius.
Therefore, transconjugante T. aromatica AR-1 R+ contained two copies of the genes for the HHQ dehydrogenase. Both genes could be expressed and could result in a thicker protein band on the SDS-PAGE. T. aromatica K 172 wt is not known to degrade any aromatic compound through the HHQ-degradation pathway, and it did not express a conspicuous protein with the mass of 50 kDa.
The properties of the 50 kDa protein agreed with the activity level, induction pattern, and localisation of HHQ dehydrogenase of A. anaerobius and T. aromatica AR-1. HHQ dehydrogenase is a membrane-bound enzyme, constitutively expressed, but up-regulated after growth with resorcinol in A. anaerobius and after growth with α-resorcylate in T. aromatica AR-1, respectively (Philipp et al., 2002). Therefore, we conclude that the 50 kDa protein encodes HHQ dehydrogenase, the second enzyme in resorcinol degradation in A. anaerobius and the first enzyme in the central pathway of HHQ degradation, respectively.
An additional protein with the mass of 42 kDa was observed only in cells grown with resorcinol. We assume that this protein represents the resorcinol hydroxylating enzyme. This correlates with biochemical studies of RH in A. anaerobius, where the specific activity of RH was measurable only after growth with resorcinol (Philipp and Schink, 1998). The other two proteins 80 and 105 kDa in mass were probably involved in further degradation of the de-aromatized HBQ, because these proteins were present in membrane fractions of A. anaerobius, and of the two transconjugantes T. aromatica AR-1 R+ and
K 172 R+ grown with resorcinol as well as in membrane fractions of wildtype T. aromatica AR-1 grown with α-resorcylate.
This article presents the newest investigations on the first two enzymes involved in the resorcinol degradation pathway. The molybdenum dependency of resorcinol hydroxylase implies a molybdenum atom in the catalytically active centre of the RH. The isolated protein with the mass of 50 kDa revealed the putative HHQ dehydrogenase, the second enzyme involved in the resorcinol degradation of A. anaerobius, but the first enzyme in the central HHQ-degradation pathway.
Acknowledgments We like to thank Dr. S. Marqués Martin for helpful advices and fruitful discussions.
6.6 References
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Blaschke, M., A. Kretzer, C. Schäfer, M. Nagel, and J.R. Andreesen (1991) Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria. Arch Microbiol 155: 164-169.
Boll, M., G. Fuchs, and J. Heider (2002) Anaerobic oxidation of aromatic compounds and hydrocarbons. Curr Opin Chem Biol 6: 604-611.
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Hellstern, J.A., B. Philipp, and B. Schink (unpublished (b)) Degradation of 2-hydroxy-1,4-benzoquinone (HBQ), the first non-aromatic intermediate in anaerobic resorcinol (1,3-dihydroxybenzene) degradation, by cell-free extracts of Azoarcus anaerobius.
Hellstern, J.A., B. Philipp, S. Marqués Martin, J. Medina Bellver, and B. Schink (unpublished (a)) Heterologous expression of the genes of Azoarcus anaerobius involved in the degradation of resorcinol (1,3-dihydroxybenzene) in Thauera aromatica strains AR-1 and K 172.
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7 Discussion
Aromatic compounds are ubiquitous in nature. They are of biological or anthropogenic origin, and are released either deliberately (e.g. herbicides) or accidentally (e.g. oil spills) into the environment. Mineralization of aromatic compounds is part of the global carbon cycle, and contributes to removal of man-made pollutants.
Degradation of aromatic compounds by microorganisms proceeds wherever essential nutrients for growth are available and abiotic conditions are appropriate. In the presence of molecular oxygen, microorganisms degrade aromatic compounds by an oxidative attack (Harwood and Parales, 1996), while in the absence of oxygen degradation of aromatic compounds is initiated by a reductive steps (Gibson and Harwood, 2002; Schink et al., 2000). An exception is found in two nitrate-reducing bacteria. Azoarcus anaerobius and Thauera aromatica AR-1 transform resorcinol (1,3-dihydroxybenzene) and α-resorcylate (3,5 dihydroxybenzoate), respectively, in peripheral pathways to hydroxyhydroquinone (HHQ, 1,2,4-trihydroxybenzene). The central intermediate HHQ is not being reduced as this was described for the sulfate-reducing bacterium Desulfovibrio inopinatus (Reichenbecher et al., 2000), but oxidized to 2-hydroxy-1,4-benzoquinone (HBQ) with nitrate as electron acceptor (Philipp and Schink, 1998 and 2000).
Resorcinol hydroxylase and nitrate reductase both are membrane-bound enzymes, and, thus, the oxidation of resorcinol could be coupled directly to the reduction of nitrate to nitrite. The redox potential of the redox pair resorcinol/
HHQ was calculated under standard conditions at pH 7 (E°´) to be -33 mV (Philipp and Schink, 1998). The released electrons could be transferred via a ubiquinone to the nitrate reductase. The terminal electron acceptor nitrate is reduced to nitrite, and energy could be generated by proton translocation. The resulting free energy under standard conditions (? G°`) is calculated to -74.7 kJ/mol. The electrons released by the dehydrogenation of HHQ to HBQ have an E°´value of +180 mV (Philipp and Schink, 1998). As this reaction is catalysed by a membrane-bound enzyme, too, the released electrons could
directly flow into the nitrate reductase system, and thereby generating a ? G°`
of -46.3 kJ/mol.
The dehydrogenation of the central intermediate HHQ, leading to the first non-aromatic compound HBQ, is an exergonic reaction. The reduction of the most prominent central intermediate during anaerobic degradation of aromatic compounds, benzoyl-CoA, depends on low-potential ferredoxin electrons and on ATP hydrolysis (Boll et al., 2002), thus, consuming instead of generating energy. If possible, nitrate-reducing bacteria should prefer such oxidation reactions. Fermenting and sulfate-reducing bacteria are incapable of performing these oxidation steps because the redox potential of their terminal electron acceptor is too low to accept electrons at this potential. The different mechanisms of resorcinol degradation demonstrate that the redox potential of the electron-accepting system determines the biochemical strategy used for degradation of aromatic compounds. This discrimination is observed also in degradation of ethylbenzene and p-cresol. While nitrate-reducing bacteria initiate degradation by molybdenum dependent hydroxylation (Kniemeyer and Heider, 2001; Hopper et al., 1991), sulfate-reducers add fumarate in order to activate the compound for further degradation (Kniemeyer et al., 2003; Müller, 2001).
In different bacteria various attempts of heterologous expression of the resorcinol hydroxylase were performed, but heterologous expression was successful only in bacteria closely related to A. anaerobius. Expression of
In different bacteria various attempts of heterologous expression of the resorcinol hydroxylase were performed, but heterologous expression was successful only in bacteria closely related to A. anaerobius. Expression of