Modular behavior of tauD provides insight into the origin of specificity in alpha-ketoglutarate-dependent nonheme iron oxygenases.
Taurine alpha-ketoglutarate dioxygenase (tauD) is one of the best-studied alpha-ketoglutarate (alphaKG)-dependent nonheme iron oxygenases. As with all oxygenases, a fine balance must be struck between generating a species sufficiently reactive for the required chemistry and controlling that species to prevent undesirable side reactions [Klinman JP (2007) Accts Chem Res 40:325-333]. In the case of tauD, the substrate oxidizing species has been shown to be a ferryl-oxo, and the introduction of deuterium at the reactive position of substrate results in an enormous kinetic isotope effect together with a partial uncoupling of oxygen activation from substrate oxidation [Price JC, Barr EW, Glass TE, Krebs C, Bollinger JM (2003) J Am Chem Soc 125:13008-13009]. We have generated a series of site-specific variants at a position that resides directly behind bound substrate (F159 to L, V, A, and G). Decreasing side-chain bulk diminishes the coupling of oxygen activation to C-H cleavage, which is further reduced by substrate deuteration. Despite this impact, oxygen activation remains completely coupled to the oxidative decarboxylation of alphaKG. The concentration of bis-Tris buffer impacts the extent of coupling of oxygen activation to C-H cleavage, implicating the buffer in the uncoupling pathway. These data indicate a critical role for residue 159 in substrate positioning and reaction in tauD and show that minor active-site perturbations in these enzymes could allow for changes in substrate reactivity while maintaining substrate triggering and oxygen binding/activation.
McCusker
Kevin P
KP
Department of Chemistry and of Molecular, University of California, Berkeley, CA 94720-1460, USA.
Klinman
Judith P
JP
D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D007656:Ketoglutaric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism; D014475:Uncoupling Agents / Q000378:metabolism
D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D013447:Sulfites; D014475:Uncoupling Agents; D019802:Succinic Acid; D007501:Iron; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C433064:taurine-alpha-ketoglutarate dioxygenase; D010100:Oxygen