Investigation of a Catalytic Zinc Binding Site inEscherichia colil-Threonine Dehydrogenase by Site-Directed Mutagenesis of Cysteine-38

The First Crystal Structure of l-Threonine Dehydrogenase

“…The molecular mass of the protein in solution determined on high performance gel chromatography (data not shown) was approximately 125,000 daltons, suggesting that this protein exists as a tetramer, as observed for TDHs from E. coli and chicken liver. [4][5][6] The crystal of PhTDH was prepared 14 but the structure of PhTDH could not be solved by molecular replacement 15 with the ADH molecule. This first reported that the structure of PhTDH was determined by the single wavelength anomalous diffraction (SAD) method, and refined to the crystallographic R and R free factors of 0.204 and 0.239 in the resolution range of 130.19 Å-2.05 Å, respectively.…”

“…3 From the structure of PhTDH, it was shown that PhTDH coordinates the zinc ion with four cysteine residues (Cys97, Cys100, Cys103 and Cys111) and the ideal tetrahedral geometry (Figure 6), as observed for human and horse liver ADHs. 20 From the high sequence homology at the lobe loop in TDHs ( Figure 1), TDHs seem to coordinate one zinc ion with four cysteine residues, as the structural zinc ion, 5 while SsADH and ApADH coordinate the zinc ion with three cysteine residues and one carboxyl residue of Glu or Asp. In the case of ApADH 12 a disulfide bond with two cysteine residues distorting the tetrahedral geometry was observed.…”

“…TDH was reported to contain one zinc ion per subunit, 26,27 but was found to be an enzyme that needs two zinc ions per subunit to exhibit activity like ADH. 5 Analysis of the metal in PhTDH indicated that it contained one zinc ion per subunit and exhibited its activity with zinc ion. 3 However, the electron density of the zinc ion was not observed at the active cleft of the crystal of PhTDH.…”

“…As observed for EcTDH, PhTDH seems to contain a functional catalytic zinc ion at the active center coordinated by Cys42, His67, Glu68 and Glu152, but the zinc ion seems to be less tightly bound at the site. 5 The active site of apo-SsADH coordinates the zinc ion with four amino acid residues (Cys38, His68, Glu69, and Cys154). 10 These four amino acids are conserved in ADHs (Figure 1).…”

Mononuclear Iron Enzymes Are Primary Targets of Hydrogen Peroxide Stress

“…CDA purified from cells is predominantly occupied by zinc with a minor amount of iron, and mechanistic studies have usually employed the zinc-loaded enzyme; however, kinetic efficiency is somewhat higher with iron (24,25). TDH exhibits good activity when constituted with zinc and is tentatively regarded as using zinc as an active-site metal in vivo; it is less active with manganese, and activity with iron has not been reported (26,27).…”