Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme Fe(II) oxygenase, catalyses the conversion of taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate and CO(2). The enzyme allows Escherichia coli to use taurine, widely available in the environment, as an alternative sulfur source. Here we describe the X-ray crystal structure of TauD complexed to Fe(II) and both substrates, alphaKG and taurine. The tertiary structure and fold of TauD are similar to those observed in other enzymes from the broad family of Fe(II)/alphaKG-dependent oxygenases, with closest structural similarity to clavaminate synthase. Using the TauD coordinates, a model was determined for the closely related enzyme 2,4-dichlorophenoxyacetate/alphaKG dioxygenase (TfdA), supporting predictions derived from site-directed mutagenesis and other studies of that biodegradative protein. The TauD structure and TfdA model define the metal ligands and the positions of nearby aromatic residues that undergo post-translational modifications involving self-hydroxylation reactions. The substrate binding residues of TauD were identified and those of TfdA predicted. These results, along with sequence alignment information, reveal how TauD selects a tetrahedral substrate anion in preference to the planar carboxylate selected by TfdA, providing insight into the mechanism of enzyme catalysis.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the alphaKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 530 and 520 nm spectra associated with alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-Fe(II)TauD species, reacts rapidly with oxygen (42 +/- 9 s(-)(1)). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by Mössbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.