TauD enzyme

Fig. 1. Construction of a computational model for TauD. (A) the solvated TauD enzyme (PDB code 1GY9, solvating water molecules in red) (B) the desolvated enzyme (C) the active site with the substrate and a-ketoglutarate bound to the iron centre, and the most important amino acids in the first and second coordination sphere (D) a minimal model for TauD including only the first coordination sphere and the substrate.

Locating minima is not always straightforward since a reaction surface is usually complex, and a geometry optimization calculation will only locate minima close to the starting point. It is usually not feasible to systematically explore all possible conformers, so chemical intuition and corroborative evidence from experiment play important roles. A nice example is the identification of the coordination geometry of oxo-iron(IV) intermediate in TauD (22). As mentioned above, during optimization of enzyme active sites, key atoms are sometimes fixed to mimic the constraints that the protein environment exerts on the active site (20). 

Scheme 2.2 Examples of reactions catalyzed by and RNA by the protein AlkB [54] (R = sugar al

The nature of the enzyme also determines the fate of the two oxygen atoms In monooxygenases (e.g. sMMO), one oxygen atom from dioxygen is transferred to the substrate while the other is reduced and forms water. In dioxygenases (e.g. TauD), both oxygen atoms are transferred to the substrate, whereas in oxidases (e.g. A9D), both oxygen atoms are reduced to water. 

Figure 11 Crystal structures of stages in the catalytic cycle of a-keto acid-dependent enzymes (a) the binary complex of CarC (lNX4.pdb), (b) the ternary complex of TauD (IGQW.pdb), and (c) the NO adduct of the ternary complex of CAS (IGVG.pdb)...

The archetype Fe(II)/aKG hydroxylase is taurine/ q KG dioxygenase (TauD), an Escherichia coli enzyme that catalyzes the conversion of taurine (2-aminoethanesulfonic acid) to sulfite and aminoacetaldehyde, as illustrated in Scheme 1. TauD catalyzes the hydroxylation of a C H bond on the carbon adjacent to the sulfonate group of taurine. The product of this reaction then decomposes to yield hydrogen sulfite, which serves as an important source of sulfur for many microorganisms. A catalytic mechanism that has been proposed for these enzymes is provided as Scheme 2. Prior to the activation and hydroxylation of the Ci carbon on taurine, q KG binds to the Fe(II) center as a chelate, displacing two of the coordinated waters. Taurine then binds to the enzyme in the vicinity of the Fe(II) center, displacing the remaining water. 

One of our goals for studying TauD was to correlate our spectroscopic results with the structural information available from the X ray crystal structure of the ternary complex prepared under anaerobic conditions. Subsequently, this information could be used as a foundation to study similarities in catalytic mechanisms between different members of this enzyme class. Because the catalytic mechanism involves the displacement of bound water molecules from the Fe(II) cofactor, we studied samples of Fe(II)NO-TauD without cosubstrates and with just the aKG added to determine if FIYSCORE could be used to follow this chemistry. Figure 16(a) shows the FIYSCORE spectrum at g = 4.00 for Fe(II)NO TauD in aqueous buffer without the two cosubstrates. Two water molecules should be coordinated to the Fe(II)NO center under these conditions. The HYSCORE shows a weak, disordered system of overlapping H arcs for this sample, which most likely reflects a high degree of... 

High-valent iron intermediates have been proposed as the active species in OAT and C-H oxidation reactions for nonheme iron enzymes. In some cases, such intermediates have been trapped by rapid fireeze-quench studies and characterized. In ribonucleotide reductase from E. coli and MMO, intermediates X and Q with Fem-( l-0)2-Ferv and Ferv-( 0,-O)2-FeIV diamond core, respectively, have been characterized (Figure 3.11).35 Also, Fe,v oxo intermediates have been observed for mononuclear proteins such as taurine/2-oxoglutarate dioxygenase (TauD) (Figure 3.11).36... 

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