1 Department of Environmental Microbiology, GBF German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
2 Department of Structural Biology, GBF German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Correspondence
Dietmar H. Pieper
dpi{at}gbf.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AJ544924 and AJ544930.
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INTRODUCTION |
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Usually, studies of regions affecting enzyme catalytic parameters have been performed by the comparison of isolates expressing different C23Os (Cerdan et al., 1995; Kitayama et al., 1996
), by the generation of chimeric proteins (Kikuchi et al., 1999
; Kitayama et al., 1996
), and/or by the selection under laboratory conditions of mutant proteins with new kinetic parameters (Cerdan et al., 1994
, 1995
; Kikuchi et al., 1999
; Wasserfallen et al., 1991
). One alternative approach to identify amino acids important to fine-tune catalytic activities is by recovering C23O natural diversity. In previous environmental studies we identified the predominant C23O gene polymorphisms in BTEX-contaminated environments (Junca & Pieper, 2003
, 2004
). Samples heavily contaminated with benzene and toluene contained an abundant C23O gene fragment (527 bp in length), identical in sequence to the C23O gene of Pseudomonas stutzeri AN10, and which differed from the gene fragment predominant in other environments by a single base (encoding a tyrosine instead of histidine at position 218), indicating that specific C23O genes and operons have been positively selected during the adaptation of soils to BTEX contamination. We also observed that out of 19 isolates harbouring such C23O enzymes, only two isolates harbouring the His218 variant could grow on benzene, toluene and ethylbenzene, whereas 17 isolates containing the Tyr218 variant could grow on benzene only. Moreover, whereas the His218 variant showed the highest activity with catechol of the substrates tested, the Tyr218 variant exhibited the highest activity with 4-methylcatechol and a relatively elevated activity with 3-methylcatechol (Junca & Pieper, 2004
). In two representative isolates, it could be shown that both the His218 and the Tyr218 variant are constitutively expressed and constitute the only catechol ring-cleavage enzyme observed in cell extracts during growth on benzene. In this work, we report the cloning, heterologous expression and kinetic characterization of these two C23O enzymes, which differ by a single amino acid position that alters enzyme function. This effect is analysed and discussed in the light of its structural and ecological significance.
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METHODS |
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PCR and molecular biology techniques.
A primer set (FER2, 5'-GCC YTG GCC TGY CRA STG TWT-3' and MUCDOCB, 5'-TTC CAG GTC ATS AGC AGY AGC GG-3') was designed based on conserved regions of C23O operon neighbouring genes (see Results). To prepare the DNA template, colonies of Pseudomonas sp. 1YB2, Pseudomonas sp. 1YdBTEX2 or recombinant E. coli strains were dissolved in 50 µl water, boiled for 10 min (Kanakaraj et al., 1998), centrifuged, and 4 µl supernatant was used as template for PCR reactions (50 µl) containing a final concentration of 1x PCR Buffer (Promega), 1·5 mM MgCl2, 200 µM each deoxyribonucleotidetriphosphate, 0·25 µM each primer (synthesized by Invitrogen) and 0·3 U µl1 Taq DNA polymerase (Promega). The PCR programme was as follows: one step at 94 °C for 5 min, 10 cycles of touchdown PCR of 94 °C for 1 min, 63 °C (1·5 °C per cycle) for 1 min and 72 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 2·5 min, followed by final elongation at 72 °C for 8 min. To determine the correct size of the amplification fragments, the PCR products were run in 1·0 % agarose gels (10 cm length, 1x TAE running buffer, 1·0 h at 95 V) and bands visualized by ethidium bromide staining (Sambrook et al., 1989
). The PCR products were cleaned with the Qiaquick PCR Cleaning Kit (Qiagen) and cloned in the pGEM-T system (Promega). The ligation products were transformed in E. coli JM109 competent cells cultured according to the manufacturer's instructions (Promega). Colonies were screened for expression of C23O by spraying with catechol (Junca & Pieper, 2004
). C23O-expressing clones were purified, and inserts from plasmids termed pC23Ohis218 and pC23Otyr218 were completely sequenced on both strands using the BigDye v1.1 system in an ABI 373A automatic DNA sequencer (Perkin-Elmer Applied Biosystems) with M13 forward and reverse primers (Sambrook et al., 1989
) and C23O inner primers described elsewhere (Junca & Pieper, 2004
). The nucleotide sequences determined in this paper can be accessed from the upgraded GenBank/EMBL/DDBJ accession numbers AJ544924 and AJ544930.
Overexpression, quantification and kinetic characterization of C23Os.
For overexpression of C23O enzymes, E. coli JM109 cells expressing C23O were grown and cell extracts prepared as described previously (McKay et al., 2003). Total protein concentrations in cell extracts were determined by the Bradford protein assay (Bradford, 1976
) with BSA as standard. For quantification of C23O proteins in cell extracts, SDS-PAGE was performed on a Bio-Rad Miniprotein II, essentially as described by Laemmli (1970)
, with acrylamide concentrations for concentrating and separating gels of 5 and 10 % (w/v), respectively. Dilutions were designed so that the amount of protein loaded ranged from 0·3 to 15 µg. Gels were incubated in a fixative solution of 10 % acetic acid/40 % ethanol for 30 min, washed twice for 30 min with 20 % ethanol and stained overnight in 0·4 µM ruthenium II tris (bathophenanthroline disulfonate) (Lamanda et al., 2004
; Rabilloud et al., 2001
). Gels were washed in 20 % ethanol/10 % acetic acid to eliminate residual matrix background staining and scanned using a Fujifilm LAS-1000 CCD camera. The fluorescence intensity was integrated, and the relative intensities of the bands corresponding to the C23O protein were determined using the AIDA 2.1 software package (Raytest Isotopenmessgeräte) (Lamanda et al., 2004
).
N-terminal sequencing and MALDI-TOF analysis were performed as previously described (Junca & Pieper, 2004).
Catalytic activities of C23O proteins were recorded on a UV 2100 spectrophotometer (Shimadzu Corporation). Activities were determined at 25 °C in 50 mM air-saturated K/Na-phosphate (pH 7·5) with catechol, 3-methylcatechol, 4-methylcatechol or 4-chlorocatechol as substrates, using the extinction coefficients of reaction products previously described (Heiss et al., 1995; Hirose et al., 1994
). One unit of enzyme activity was defined as the amount of enzyme that forms 1 µmol product per minute. Specific activities are expressed as units per gram of protein. C23O proteins utilize a compulsory-order ternary complex mechanism in which binding of the catecholic substrate precedes that of oxygen. Kinetic data were recorded in experiments where only one of the substrates was varied. Vmax, kcat and apparent KmA values (
) for catecholic substrates were determined using 1100 µM substrate in air-saturated buffer, and kinetic data were calculated from the initial velocities using the MichaelisMenten equation by non-linear regression (KaleidaGraph, Synergy Software). As very low
values were indicated by this method, kinetic data were finally determined from progress curves obtained from reactions with initial substrate concentrations of 10 µM. The catalytic rate at each time point, v(t), was obtained by the photometer software from the slope of the absorbance change:
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The substrate concentrations S(t) at each time point were calculated by the amount of ring-cleavage product formed during the further course of the reaction:
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The apparent for oxygen (
) was determined from progress curves obtained from reactions with initial concentrations of catecholic substrates of 300 µM and 5060 µM oxygen. The buffer was made anaerobic by alternate vacuum treatment and purging with nitrogen. Eight hundred microlitres of this buffer in 1 ml stoppered cuvettes was gently mixed with 200 µl air-saturated buffer containing 1·5 mM catecholic substrate. As previously described (Nakajima et al., 2002
), the concentration of oxygen in a reaction mixture can be quantified very sensitively based on extradiol cleavage of catecholic substrates. Thus, the total change of absorbance after addition of C23O is a measure of the amount of oxygen present in the reaction. With catechol as substrate, a rapid rise in absorbance of 1·82·0 was observed under the above reaction conditions, indicating the formation of 5055 µM 2-hydroxymuconic semialdehyde. Back-diffusion of oxygen was negligible under the experimental conditions. The catalytic rate at each time point, v(t), was obtained by the photometer software from the slope of the absorbance change, as described above. Oxygen concentrations during the reaction were calculated by the amount of ring-cleavage product formed during the further course of the reaction as described above with:
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The enzyme concentrations used (5080 nM) ensure that less than 20 % of the enzyme was inactivated during the course of the reaction. The datasets obtained were fitted to the MichaelisMenten equation by non-linear regression (KaleidaGraph, Synergy Software).
Partition ratios (the number of substrate molecules consumed per number of enzyme molecules inactivated) of C23OHis218 and C23OTyr218 were determined under saturating substrate concentrations (100 µM). The amount of enzyme (approx. 5 nM) was such that the enzyme was completely inactivated before 20 % of the substrate was consumed.
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RESULTS |
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Cell extracts of E. coli JM109 (pC23Ohis218) expressing C23OHis218 and E. coli JM109 (pC23Otyr218) expressing C23OTyr218 were analysed by SDS-PAGE, and a prominent band of 35±2 kDa, which was absent in cell extracts of E. coli JM109 (pGEM-T), was observed (Fig. 1). The identity of the bands with C23OHis218 and C23OTyr218 was confirmed by N-terminal sequencing and MALDI-TOF analysis. The relative amounts of these C23O protein bands were 29±2 % of the total protein content in each case, showing that expression levels of the two C23O variants did not differ significantly (Fig. 1
). As C23Os are known to be subject to inactivation during protein purification due to oxidation and/or loss of active site ferrous iron (Eltis et al., 1993
; McKay et al., 2003
), kinetic data were measured directly in these cell extracts. Spontaneous inactivation in these extracts was negligible: <10 % in 8 h. Moreover, spontaneous oxygen consumption was negligible, such that no interference with kinetic measurements can be expected.
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C23O enzymes are characterized by their sometimes rapid inactivation through oxidation of active site iron during catalytic turnover. As an example, the partition ratio of C23Omt-2 with 4-ethylcatechol was reported to be 6500, whereas that of 3-methylcatechol was 210 000 (Cerdan et al., 1994). Whereas there was no significant difference in partition ratio between the variants (Table 1
), the partition ratios observed with all three substituted catechols analysed were dramatically lower than those observed for C23Omt-2.
The affinity for oxygen is another parameter critical to the activity of C23Os. Both enzymes exhibited values of 810 µM in the presence of saturating concentrations of catechol (Table 1
). These values are slightly higher than those reported for C23Omt-2 (3·9 µM) (Kukor & Olsen, 1996
).
values for oxygen in the presence of saturating concentrations of 3-methyl-, 4-methyl- or 4-chlorocatechol varied only slightly (by a factor of 2) in the case of C23OTyr218. However, in comparison, values for oxygen in the presence of saturating concentrations of 4-chlorocatechol and 3-methylcatechol were significantly increased in the case of C23OHis218.
Evidently, the nature of the amino acid at position 218 has a prominent effect on the enzyme kinetics. In previously described C23Os, this position is usually occupied by histidine or phenylalanine and, exceptionally, leucine (Fig. 2). A tyrosine in this position was only observed in a thermostable chimeric gene, which contained a central C23O gene fragment from an environmental sample (Okuta et al., 1998
). This position was, however, never considered relevant for enzyme functioning. To understand possible influences of histidine/tyrosine variants on enzyme function, three-dimensional models of the two protein types were constructed, based on the available crystal structure of the homologous and highly similar (identities 290/307, 94 %) XylE protein (C23Omt-2) (Kita et al., 1999
) (Fig. 3
). Amino acid 218 is not involved in the formation of the substrate-binding pocket, nor does it interfere with the hydrophobic channel through which catechols and dioxygen are thought to enter the active site (Kita et al., 1999
). This amino acid is localized on the side of the molecule, which is buried in the tetramer interface, and appears to be part of a second smaller channel leading to the active centre. The functional role of this channel (Fig. 3
) is unclear, and His/Tyr218, with a distance of more than 9 Å to the active iron and pointing away from it, is unlikely to interfere much with water or oxygen molecules filling the channel.
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DISCUSSION |
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Interestingly, several characteristics distinguish the naturally occurring Tyr218 and His218 variants. The Tyr218 variant, detected both in highly and slightly contaminated soil, and present in benzene degraders, exhibits low turnover number and high affinity, whereas the His218 variant, detected in highly contaminated sites only, and present in toluene/benzene degraders, exhibits a high turnover number and low affinity. As such, the variants seem to be selected for by the environmental conditions. Whether the different growth phenotypes of the strains harbouring the Tyr218 and His218 variants are actually a consequence of the different kinetic characteristics of the enzymes or result from other characteristics of the host will need to be analysed by site-directed mutagenesis in the same strain.
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ACKNOWLEDGEMENTS |
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Received 30 June 2004;
revised 23 August 2004;
accepted 27 August 2004.
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