Characterization of three XylT-like [2Fe-2S] ferredoxins associated with catabolism of cresols or naphthalene: evidence for their involvement in catechol dioxygenase reactivation

Abstract

The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

FIG. 1

Sequence alignment of the four ferredoxins considered in the present study. Sequences were aligned using the GeneWorks software (version 2.5N) from IntelliGenetics. Identical residues are shaded. Cysteines presumed to serve as ligands for the [2Fe-2S] cluster are indicated in boldface. The sources, accession numbers, and references for each sequence are as follows: XylT-MT2, XylT from P. putida mt-2 (M64747) (8); DmpQ-CF600, DmpQ from Pseudomonas sp. strain CF600 (X60835) (28); PhhQ-P35X, PhhQ from P. putida P35X (X79063) (23); and NahT-KF715, NahT from P. putida sp. strain KF715 (S78585) (22). The XylT sequence was modified to take into account a correction of the gene sequence resulting in an alanine-to-glycine replacement of residue 76 (12).

FIG. 2

SDS-PAGE analysis of purified DmpQ, NahT, PhhQ, and XylT. The proteins were purified as described in Materials and Methods and analyzed (1.0 μg per well) by electrophoresis on a 16% polyacrylamide gel containing 10% glycerol in a Tris-Tricine system. Sizes of protein markers (lane M) are indicated.

FIG. 3

EPR spectra of the XylT-like ferredoxins. Purified samples of DmpQ (134 μM), PhhQ (133 μM), and NahT (96 μM) were reduced with 1 mM dithionite. Spectra were recorded at 10 K under the following conditions: microwave power, 0.01 mW at 9.65 GHz; modulation amplitude, 1 mT at 100 kHz. The inset shows spectra of PhhQ (110 μM) and NahT (70 μM) samples containing 16% glycerol.

FIG. 4

Time course of ferredoxin-dependent XylE reactivation. A preparation of 4-methylcatechol-inactivated XylE (0.1 μM) was incubated in 0.2 ml of reactivating buffer (0.1 mM potassium phosphate [pH 7.5], 2.5 μM 5-deazaflavin, 10 mM glycine) containing the indicated ferredoxin at 0.5 μM. Reactivation was initiated by light. XylE activity was assayed on a 15-s time scale and expressed as a percentage of the maximal activity measured at 5 min in the XylT-mediated reactivation assay (100% = 120 U/mg).

FIG. 5

Cross-linking of XylE and XylT analogues. XylE (3.6 μM) was allowed to react with XylT, DmpQ, PhhQ, or NahT (20 μM) in the presence of EDC as described in Materials and Methods. Samples were analyzed by SDS-PAGE on a 12% polyacrylamide gel (lanes 2, 4, 6, and 8). The samples analyzed in lanes 3, 5, 7, and 9 were from cross-linking reactions performed in the presence of 0.1 M NaCl. Lane 1 was loaded with a mixture of unreacted XylE and XylT. The arrow indicates the cross-linked 46-kDa product.