Affiliation:
1. Fachbereich Biologie, Universität Konstanz, 78457 Constance, Germany
Abstract
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 H
2
O to acetylene (H—C≡C—H) to form acetaldehyde (CH
3
CHO). 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
43 articles.
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