title: Exploring the active site of the tungsten, iron-sulfur enzyme acetylene hydratase.
abstract: The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H₂O to acetylene (H-C≡C-H) to form acetaldehyde (CH₃CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.
authors.affiliation: Universität Konstanz, Fachbereich Biologie, Constance, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Tenbrink
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schink
authors.forename: Peter M H
authors.initials: PM
authors.lastname: Kroneck
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D020564:Deltaproteobacteria / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D014414:Tungsten / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D006836:Hydro-Lyases; C096200:acetylene hydratase; D014414:Tungsten