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  1. The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases. 
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  4. Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (Chd T ), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. Chd T forms a “head-to-tail” homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His 117 , His 257 , Asp 116 , Asn 216 , and a water/hydroxide as ligands. A conserved His residue, His 114 , is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π–π interaction with Trp 227 . On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation. 
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