Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Ap. 1095, 41080 Sevilla, Spain
Correspondence
Eduardo Santero
esansan{at}dex.upo.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Full-colour versions of Figs 2 and 3 are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
Present address: Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, Ctra Utrera Km 1, 41013 Sevilla, Spain.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because of their importance in degradation of aromatic rings, extradiol dioxygenases have received a lot of attention. A large number of genes encoding extradiol dioxygenases have been sequenced from many different bacteria. Sequence and crystallographic data suggest that these enzymes arose from at least two different evolutionary origins. Most of them are type I enzymes, which contain the PROSITE extradiol dioxygenase fingerprint, whilst a few, represented by LigAB, cannot be aligned with type I enzymes and are grouped as type II (Eltis & Bolin, 1996; Sugimoto et al., 1999
). In spite of the phylogenetic divergence, all types of extradiol dioxygenases appear to have similar catalytic mechanisms (Bugg & Lin, 2001
; Que & No, 1996
; Solomon et al., 2000
), which involve non-haem Fe(II) in the active site. The role of some strictly conserved residues as metal ligands or active-site residues may be inferred from structural and biochemical data (Han et al., 1995
; Senda et al., 1996
, 1997
; Shu et al., 1995
; Uragami et al., 2001
). However, even for the best-characterized enzymes, the 2,3-dihydroxybiphenyl dioxygenases (DHBDs), the reaction mechanism has not been fully established, nor has the exact role of some active-site residues. Of course, the role of other conserved residues, which may be relevant for enzyme function or substrate specificity, has not been characterized either.
Within the two-domain extradiol dioxygenases bearing the catalytic site in the C-terminal domain, sequence comparison shows that evolutionary relationships correlate with specificity of substrate. These enzymes can be divided in two separate clades: those showing preference for monoaromatic compounds and those showing preference for polycyclic substrates (Eltis & Bolin, 1996; Harayama & Rekik, 1989
). More recent sequence comparison revealed that, among enzymes cleaving polycyclic compounds, 1,2-dihydroxynaphthalene dioxygenases (DHNDs) cluster together within the subfamily I.3.E, and that two groups within this subfamily can be established (Andújar et al., 2000
). Members of this subfamily of DHNDs are less well characterized and biochemical data are not available or are very scarce for many of them. They share a number of conserved residues but it is not known whether this conservation is functionally important or merely reflects a common evolutionary origin.
The extradiol dioxygenase ThnC, required for biodegradation of the organic solvent tetralin by Sphingomonas sp. strain TFA, has been recently purified and characterized (Andújar et al., 2000). It catalyses the extradiol cleavage in a position proximal to the alicyclic ring of tetralin (Fig. 1
). Alignment of its deduced amino acid sequence with those of other two-domain enzymes showed that ThnC lies within group 2 of DHNDs (Andújar et al., 2000
) (Fig. 2
), a group composed of enzymes able to transform a broad range of substrates, some of which were in fact initially identified as DHBDs. Biochemical properties of the enzyme have been fully characterized. Like many other extradiol dioxygenases (Adams et al., 1992
; Asturias & Timmis, 1993
; Happe et al., 1993
; Kim & Zylstra, 1995
), ThnC shows a very evident inhibition by the substrates tested, although it is particularly sensitive to inhibition by the catechol intermediate of the tetralin biodegradation pathway, 1,2-dihydroxytetralin (1,2-DHT) (Andújar et al., 2000
). Activity of the enzyme against different substrates showed that, as its sequence suggested, ThnC is a true DHND, although it can also efficiently transform 1,2-DHT and 2,3-dihydroxybiphenyl (2,3-DHB) (Andújar et al., 2000
).
|
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An 830 bp ApaIHindIII fragment from pIZ592 (Andújar et al., 2000) was cloned between the ApaI and HindIII sites of the multiple cloning sequence of pBluescript II SK(+) (Stratagene) to yield pIZ593. Site-directed mutagenesis to construct the mutations V175D, Q198D, N213H, A251M, A282R and A282P was performed following the method of Kunkel et al. (1987)
, using the mutagenic oligonucleotides shown in Table 1
. To construct the mutations Y177I, Q198H, G206M and A282G, site-directed mutagenesis was performed by PCR (Ho et al., 1989
) using a pair of complementary mutagenic oligonucleotides (Table 1
). The mutations were confirmed by sequencing, and the mutated ApaIHindIII fragments of 830 bp were used for substitution of the wild-type sequence in the plasmid pIZ591, a plasmid bearing a His-tag fused to thnC (Andújar et al., 2000
).
|
Enzyme assay.
Since specific activity of different preparations of ThnC may vary because of oxidation or loss of Fe2+ during purification (Andújar et al., 2000), all purified enzymes were reactivated by incubation on ice with 2 mM ammonium ferrous sulfate for 20 min, immediately before any enzyme assay.
One unit of enzyme activity was defined as the amount of enzyme that converts 1 µmol substrate min-1. Cell extracts or purified protein were added to a solution (final volume, 1 ml) containing 50 mM sodium/potassium phosphate buffer (pH 6·8) and 25 µM 1,2-DHT at 25 °C. 1,2-DHND activity was assayed by measuring substrate consumed in 50 mM acetate buffer (pH 5·5), as previously described (Kuhm et al., 1991), using the following absorption coefficient:
max=331 nm,
=2·60 mM-1 cm-1. Extradiol dioxygenase activity with other substrates was assayed by measuring the formation of the corresponding ring-fission products. The following absorption coefficients were used (in 50 mM sodium/potassium phosphate buffer pH 6·8): 1,2-DHT (Andújar et al., 2000
),
max=336 nm,
= 2·26 mM-1 cm-1; 2,3-DHB (Eltis et al., 1993
),
max=434 nm,
=13·20 mM-1 cm-1. Protein concentration was determined by the method of Bradford (1976)
, with bovine serum albumin as the standard. All colorimetric assays were quantified using a Beckman DU 640 spectrophotometer.
The ranges of substrate concentrations used in enzyme assays for determination of the kinetic parameters were: [1,2-DHT]=5250 µM; [2,3-DHB]=7·5 µM4 mM; [1,2-dihydroxynaphthalene (1,2-DHN)]=10300 µM.
To perform enzyme assays at different pHs, the following buffers were used at a concentration of 50 mM: citric acid pH 4·0, pH 4·5 and pH 5·0; MES pH 5·5 and pH 6·0; PIPES pH 6·5 and pH 7·0; TRIZMA pH 7·6, pH 8·0, pH 8·5 and pH 9·0. Absorption coefficients of 1,2-DHT at different pHs were previously calculated (Andújar et al., 2000).
Sequence analysis comparison and structural model.
The amino acid sequence of ThnC was initially compared to those in the databases using the BLASTp program (Altschul et al., 1997). Amino acid sequences which showed high similarity to that of strain TFA were aligned using the CLUSTAL W program (Thompson et al., 1994
) using default parameters.
The computer program Swiss PDB Viewer (Guex & Peitsh, 1997) was used to build the structural model of ThnC using the crystal coordinates of BphC from Burkholderia fungorum LB400 (Han et al., 1995
) as the reference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The structural model of ThnC using the crystal coordinates of BphC of LB400 as a reference (Han et al., 1995) showed a good fit with that of BphC and revealed the conservation of the two structural domains and the two motifs
in the catalytic C-terminal domain. The three-dimensional model (Fig. 3
) showed that each of the two additional
-sheets at the C-terminal end may interact with each
motif of the C-terminal domain, as shown for the two crystallized BphC enzymes (Han et al., 1995
; Senda et al., 1996
; Uragami et al., 2001
).
|
Mutagenesis of thnC and purification of the modified gene products
In order to test the relevance of the residue identity in the selected positions for ThnC activity, the C-terminal domain of ThnC was modified. To this end, 10 substitutions were made on a DNA fragment consisting of the C-terminal region of thnC. The substitutions, together with information about the residues in equivalent positions of different extradiol dioxygenases, are summarized in Table 1. Mutations were confirmed by sequencing. The mutated fragments were used for substitution of the wild-type sequence in the plasmid pIZ591, which contains thnC His-tagged at its N-terminus, and expressed from the T7 promoter (Andújar et al., 2000
).
The resulting plasmids, together with pIZ591, were used to transform the overproducing strain NCM631/pIZ227 (Govantes et al., 1996). After induction of the transformed strains with IPTG, overproduction was very evident in all cases. In spite of the different degrees of solubility of the His-tagged proteins, all were successfully purified in a single step by affinity chromatography, and preparations showed a single band after electrophoresis (data not shown).
pH range of of the mutant proteins
Because the activity assays using 1,2-dihydroxynaphthalene (1,2-DHN) as the substrate had to be carried out at pH 5·5 (Kuhm et al., 1991), preliminary experiments aimed at characterization of mutant proteins included tests of activity at different pH values, using the substrate for ThnC intermediate in the tetralin biodegradation pathway, 1,2-DHT. Activity of wild-type ThnC showed a strong dependence on pH with an optimum at pH 6·87, and just 3540 % of the activity at pH 5·5. Some mutant proteins exhibited activity significantly more tolerant to acidic pH, although their pH optima were the same as that of wild-type ThnC. Activities of these proteins at pH 5·5 relative to their activities at pH 6·8 were the following: 62 % for N213H, 69 % for G206M or A282R, and 65 % for A282G. Strikingly, mutant Y177I exhibited a sharp peak of activity as a function of pH but the optimum pH was displaced from 7·0 to 5·5. Because of this, subsequent characterization of the activity of this mutant was always carried out at pH 5·5, regardless of the substrate used.
Kinetic parameters of the mutant proteins
Activity of each enzyme was assayed at different concentrations of 1,2-DHT. All enzymes exhibited strong activity inhibition at high substrate concentrations. This phenomenon was previously described for ThnC (Andújar et al., 2000) and for other extradiol dioxygenases (Kim & Zylstra, 1995
; Kuhm et al., 1991
; Kunkel et al., 1987
; La Du et al., 1958
). The kinetic data fitted well with the polynomial equation [S]/V=Km/Vmax+(1/Vmax) [S]+(1/Kss.Vmax) [S]2 previously described for enzymes showing substrate inhibition and it was therefore used to calculate the theoretical kinetic parameters from the empirical data. Kinetic parameters together with the observed activities at the optimum substrate concentration (Vobs) are shown for all mutant enzymes in Table 2
.
|
Three other substitutions, N213H, Q198H and G206M, produced enzymes with significantly higher rates of substrate turnover (kcat at least twofold higher than that of wild-type ThnC), which suggests that residues in these positions also contribute to efficient catalysis of 1,2-DHT cleavage. Of these, G206M was the mutation that most increased substrate affinity, as evidenced by its Km, in addition to the increase in turnover rate. Interestingly, the enzyme bearing this substitution was also more sensitive to substrate inhibition.
The remaining substitutions, Q198D, V175D, Y177I and A251M, had negative effects on the rate of 1,2-DHT turnover. The effects, though significant, were weak and none of the substitutions reduced the enzyme activity more than 50 %. On the other hand, Q198D and V175D significantly increased affinity for 1,2-DHT (Km approximately half of that of wild-type ThnC), thus compensating their lower turnover rate (see their kcat/Km).
Substrate specificity of the mutant proteins
In order to determine whether the effects of the substitutions on the activity towards 1,2-DHT were general, or specific to the substrate used in the reaction, activity of each enzyme was also tested using different concentrations of 2,3-DHB and 1,2-DHN. Fig. 4 shows the specificity of each protein for each substrate tested. Specificity for 1,2-DHT or 2,3-DHB is shown at the optimum pH of each protein (pH 5·5 for Y177I and pH 6·8 for all the other protein variants). Activity using 1,2-DHN as the substrate was always tested at pH 5·5.
|
The substitution G206M increased threefold the specificity for 1,2-DHT but its specificity for other substrates was very slightly reduced. Therefore, this replacement resulted in an extradiol dioxygenase particularly more specific for the intermediate of the tetralin degradation pathway.
The other two substitutions which increased specificity for 1,2-DHT, Q198H and N213H, also had a positive effect when using any of the other substrates, thus showing that histidines at these positions improve the functionality of ThnC regardless of the substrate to be transformed.
A251M increased specificity for 2,3-DHB while slightly reducing specificity for 1,2-DHT and 1,2-DHN, thus resulting in a protein particularly more specific for 2,3-DHB. On the other hand, V175D showed very slightly increased specificity for 1,2-DHT but significantly lower specificity for the other substrates. The two replacements Q198D and Y177I did not show significant effects which were dependent on the substrate transformed.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ThnC showed significantly high activity towards 1,2-DHT [24 U (mg protein)-1], the substrate in the tetralin biodegradation pathway, and also towards 1,2-DHN and 2,3-DHB, the substrates in the naphthalene or the biphenyl biodegradation pathways (Table 2, Fig. 4
). The activity towards 2,3-DHB, 7 U (mg protein)-1, is comparable to or higher than activities shown by other DHBDs. For instance, BphC1 and BphC2 from Sphingomonas sp. strain BN6 showed 3·8 and 6·57 U (mg protein)-1, respectively (Heiss et al., 1995
, 1997
). This suggests that ThnC could also be efficiently used for degradation of 2,3-DHB. In spite of these data, 5 out of the 10 mutant enzymes showed at least twofold higher specificity for 1,2-DHT (Fig. 4
), thus indicating that activity of ThnC is not maximized for its involvement in tetralin biodegradation, and, therefore, that development of an improved catalyst for 1,2-DHT oxidation is feasible.
Asn-213 in ThnC was substituted by His, a residue conserved in equivalent positions of extradiol dioxygenases of all families with the exception of those clustering within the I.3.E subfamily of DHNDs. Substitution resulted in a significantly more active enzyme using any of the substrates tested, thus indicating that His in this position may be important for activity of extradiol dioxygenases, presumably by influencing the environment of the Fe(II)-binding site, since it forms a hydrogen bond with the metal-binding His-210 in LB400 (Han et al., 1995). Similarly, Gln-198 in ThnC was substituted by His, a residue conserved in enzymes cleaving polycyclic substrates not belonging to the subfamily of DHND, which forms a hydrogen bond with the putative O2-binding His-195 in BphC from LB400 (Vaillancourt et al., 2002
). Substitution also increased activity of ThnC towards any of the substrates tested. The effects of the N213H and Q198H substitutions clearly show the relevance of His residues in these positions for activity of ThnC, which is fully consistent with their conservation among extradiol dioxygenases not belonging to the I.3.E subfamily. It is, therefore, to be expected that catalysis by DHNDs in general may be improved by changing the residues in these positions to His. On the other hand, these results are intriguing since there is no obvious explanation for the absence of these histidines among DHNDs (Fig. 2
). It is unlikely that absence of these His in DHNDs merely reflects a common evolutionary origin from an ancestor lacking them; their absence might therefore confer some particular characteristics upon DHNDs which are not observable in our assays.
Substitution A251M increased ThnC specificity for 2,3-DHB but reduced its specificity for 1,2-DHT and 1,2-DHN, thus resulting in a protein threefold more specific for 2,3-DHB, in comparison to the other substrates, than wild-type ThnC (Fig. 4). This result indicates that identity of the residue at this position may affect the activity towards a particular substrate, which confirms the interpretation of crystallographic data suggesting that this position (246 in BphC from LB400) could help to define the substrate-binding site (Han et al., 1995
). The effect of this substitution may also explain the high degree of conservation of Met in this position among enzymes cleaving 2,3-DHB (Fig. 2
).
Although the position equivalent to 206 of ThnC is conserved within the two groups of the I.3.E subfamily of DHNDs, it is not particularly conserved among other members of the family I.3 (Fig. 2), which suggests that this position tolerates a number of residues. However, substitution G206M, to give the Met residue conserved in DHNDs of group 1, increased specificity particularly for 1,2-DHT (Fig. 4
). This shows that identity of the residue in this position also may affect activity towards a particular substrate, at least within the context of ThnC, in agreement with crystallographic data, which indicated that this position surrounds the entranceway of the substrate (Senda et al., 1996
). On the other hand, since the activity towards 1,2-DHN or 2,3-DHB is not significantly altered by this substitution, the fact that DHNDs of group 1 contain Met in this position whilst those of group 2 contain Gly does not explain the higher specificity of group 2 enzymes for 2,3-DHB.
The position equivalent to 282 of ThnC is conserved within the two groups of the subfamily of DHNDs (Fig. 2). Substitutions in this position, the first in the most C-terminal
-sheet (
R in Fig. 2
), revealed its importance for activity of ThnC. On the one hand, the A282P substitution resulted in a virtually inactive protein. Crystallographic data indicated that this C-terminal
-sheet interacts with the last
motif of the protein. Since Pro provides rigidity to the protein backbone, lack of activity of the mutant protein may be explained by wrong orientation of the C-terminal
-sheet, which could prevent interaction with the last
motif, thus showing the importance of this interaction for functionality of ThnC. On the other hand, the other two substitutions, A282R and A282G, increased specificity of ThnC for 1,2-DHT, while affecting very little the specificity for the other substrates (Fig. 4
). This clearly shows that identity of the residue in this position may specifically affect catalysis of the transformation of particular substrates, and therefore, catalysts with different substrate specificities might be developed from DHNDs by changing the residue at this position. It is interesting to note that this and neighbouring positions are not particularly well conserved among extradiol dioxygenases other than DHNDs and that some of them contain Pro in this position. These data suggest that the importance of the identity of the residue in this position shown by the mutational analysis may only be pertinent for DHNDs.
Mutational analysis of ThnC has, therefore, shown that histidines in positions 198 and 213 improve functionality of the protein, consistent with their conservation among extradiol dioxygenases of other subfamilies, that the most C-terminal -sheet is important for activity and substrate specificity, which had not been shown before, and that identity of the residues in two additional positions, 206 and 251, may also affect catalysis of particular substrates.
Another observation is that some substitutions altered the sensitivity of the enzyme activity to pH, resulting in enzymes more tolerant to low pH. The most dramatic effect was shown by the substitution Y177I, which showed maximal activity at pH 5·5. At pH 5·5, the imidazole ring of the metal-binding His, and other potentially important His residues, may be protonated. This has been used to explain loss of activity of BphC from KKS102 below pH 6, since protonated His may easily lose the ability to coordinate to Fe(II) (Senda et al., 1996). The effect of the substitution Y177I may be interpreted as loss of enzyme activity at pH higher than 5·5, since its maximal activity is not higher than that of wild-type ThnC at this pH. However, the high relative activity shown by wild-type ThnC (40 % of its activity at neutral pH) and the even higher relative activity shown by some mutants (up to 70 % of their activity at neutral pH) clearly indicate that extradiol dioxygenases may be active at this acidic pH, possibly because the pKa of the essential His residues may be influenced by vicinal residues.
Extradiol dioxygenases show inhibition by the substrate but the mechanism of this inhibition is not known. It has been proposed that the substrate might be involved in negative cooperativity of the subunits, although it is unlikely, since this inhibition has also been reported for a monomeric enzyme (Happe et al., 1993). An alternative explanation could be that the substrate binds to a second site, maybe in the N-terminal domain, and this has a negative allosteric effect on activity. However, substitutions V175D and Q198D, which significantly altered the Km of the enzyme, also altered sensitivity to substrate inhibition (Table 2
), thus indicating that inhibition is apparently exerted through the same binding site, as previously suggested by kinetic analysis of the DHBD from LB400 (Vaillancourt et al., 1998
). A possible mechanism for the substrate inhibition would be that high concentration of the substrate at the entranceway might jam the enzyme, thus preventing proper fitting of the substrate in the active site, or release of the product after catalysis.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Madden, T. L., Schaffer A. A. , Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 33893402.
Andújar, E., Hernáez, M. J., Kaschabek, S. R., Reineke, W. & Santero, E. (2000). Identification of an extradiol dioxygenase involved in tetralin biodegradation: gene sequence analysis and purification and characterization of the gene product. J Bacteriol 182, 789795.
Asturias, J. A. & Timmis, K. N. (1993). Three different 2,3-Dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6. J Bacteriol 175, 46314640.[Abstract]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Bugg, T. D. H. & Lin, G. (2001). Solving the riddle of the intradiol and extradiol catechol dioxygenases: how do enzymes control hydroperoxide rearrangements? Chem Commun 2001, 941952.[CrossRef]
Eltis, L. D. & Bolin, J. T. (1996). Evolutionary relationships among extradiol dioxygenases. J Bacteriol 178, 59305937.[Abstract]
Eltis, L. D., Hofmann, B., Hecht, H. J., Lunsdorf, H. & Timmis, K. N. (1993). Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem 268, 27272732.
Govantes, F., Molina-López, J. A. & Santero, E. (1996). Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae. J Bacteriol 178, 68176823.[Abstract]
Guex, N. & Peitsh, M. C. (1997). SWISS-MODEL and the Swiss-Pdb viewer: an environment for comparative protein modeling. Electrophoresis 18, 27142723.[Medline]
Han, S., Eltis, L. D., Timmis, K. N., Muchmore, S. W. & Bolin, J. T. (1995). Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad. Science 270, 976980.[Abstract]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557580.[Medline]
Happe, B., Eltis, L. D., Poth, H., Hedderich, R. & Timmis, K. N. (1993). Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. J Bacteriol 175, 73137320.[Abstract]
Harayama, S. & Rekik, M. (1989). Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. J Biol Chem 264, 1532815333.
Heiss, G., Stolz, A., Kuhm, A. E, Müller, C., Klein, J., Altenbuchner, J. & Knackmuss, H.-J. (1995). Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6. J Bacteriol 177, 58655871.[Abstract]
Heiss, G., Müller, C., Altenbuchner, J. & Stolz, A. (1997). Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad. Microbiology 143, 16911699.[Abstract]
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 5159.[CrossRef][Medline]
Kim, E. & Zylstra, G. (1995). Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp. strain B1. J Bacteriol 177, 30953103.[Abstract]
Kuhm, A. E., Stolz, A., Ngai, K. & Knackmuss, H. J. (1991). Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids. J Bacteriol 173, 37953802.[Medline]
Kunkel, T., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-directed mutagenesis without phenotypic selection. Methods Enzymol 154, 367382.[Medline]
La Du, B. N., Zannoni, V. G., Laster, L. & Seegmiller, J. E. (1958). The nature of the defect in tyrosine metabolism in alkaptonuria. J Biol Chem 230, 251260.
Que, L. & Ho, R. Y. N. (1996). Dioxygen activation by enzymes with mononuclear non-heme iron active sites. Chem Rev 96, 26072624.[CrossRef][Medline]
Schwarcz, R., Okuno, E., White, R. J., Bird, E. D. & Whetsell, W. O., Jr (1988). 3-Hydroxyanthranilate oxygenase activity is increased in the brains of Huntington disease victims. Proc Natl Acad Sci U S A 85, 40794081.[Abstract]
Senda, T., Sugiyama, K., Narita, H., Yamamoto, T., Kimbara, T., Fukuda, M., Sato, M., Yano, K. & Mitsui, Y. (1996). Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. J Mol Biol 255, 735752.[CrossRef][Medline]
Senda, T., Sugimoto, K., Ishikazi, T., Yamada, T., Okano, M., Masai, E., Fukuda, M. & Mitsui, Y. (1997). Structural studies on the "BphC" enzyme a non-heme iron dioxygenase from a Pseudomonas sp. J Inorg Biochem 67, 336.[CrossRef]
Shu, L., Chiou, Y. M., Orville, A. M., Miller, M. A., Lipscomb, J. D. & Que, L. Jr (1995). X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 24, 66496659.
Solomon, E. I., Brunold, T. C., Davis, M. I. & 7 other authors (2000). Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem Rev 100, 235350.[CrossRef][Medline]
Sugimoto, K., Senda, T., Aoshima, H., Masai, E., Fukuda, M. & Mitsui, Y. (1999). Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions. Structure Fold Des 7, 953965.[CrossRef][Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Uragami, Y., Senda, T., Sugimoto, K., Sato, N., Nagarajan, V., Masai, E., Fukuda, M. & Mitsui, Y. (2001). Crystal structure of substrate free and complex forms of reactivated BphC, an extradiol type ring-cleavage dioxygenase. J Inorg Biochem 83, 269279.[CrossRef][Medline]
Vaillancourt, F. H., Han, S., Fortin, P. D., Bolin, J. T. & Eltis, L. D. (1998). Molecular basis for the stabilization and inhibition of 2,3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol. J Biol Chem 273, 3488734895.
Vaillancourt, F. H., Barbosa, C. J., Spiro, T. G., Bolin, J. T., Blades, M. W., Turner, R. F. & Eltis, L. D. (2002). Definitive evidence for monoanionic binding of 2,3-dihydroxybiphenyl to 2,3-dihydroxybiphenyl 1,2-dioxygenase from UV resonance Raman spectroscopy, UV/Vis absorption spectroscopy, and crystallography. J Am Chem Soc 124, 24852496.[CrossRef][Medline]
Received 3 October 2002;
revised 25 February 2003;
accepted 28 February 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |