Identification of protein fold and catalytic residues of gamma-hexachlorocyclohexane dehydrochlorinase LinA.
gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level and for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of proteins containing scytalone dehydratase, 3-oxo-Delta(5)-steroid isomerase, nuclear transport factor 2, and the beta-subunit of naphthalene dioxygenase-all known structures with different functions. The catalytic and the active site residues of LinA are predicted on the basis of its homology model. Nine mutants that carry substitutions of the proposed catalytic residues were constructed by site-directed mutagenesis. In addition to these, eight mutants that have a potential to make contact with the substrate were prepared by site-directed mutagenesis. These mutants were expressed in Escherichia coli, and their activities in crude extract were evaluated. Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity. We conclude that LinA catalyzes the proton abstraction via the catalytic dyad H73-D25 by a similar mechanism as described for scytalone dehydratase. The results suggest that LinA and scytalone dehydratase evolved from a common ancestor. LinA may have evolved from an enzyme showing a dehydratase activity.
Nagata
Y
Y
Graduate School of Life Sciences, Tohoku University, Sendai, Japan. aynaga@ige.tohoku.ac.jp
Mori
K
K
Takagi
M
M
Murzin
A G
AG
Damborský
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001673:Biodegradation, Environmental; D020134:Catalytic Domain / Q000235:genetics*; D049308:Dioxygenases; D019143:Evolution, Molecular; D006836:Hydro-Lyases; D008190:Lyases; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D029543:Nucleocytoplasmic Transport Proteins; D010105:Oxygenases; D017510:Protein Folding; D016415:Sequence Alignment; D020578:Sphingomonas / Q000201:enzymology; D013251:Steroid Isomerases
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D029543:Nucleocytoplasmic Transport Proteins; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; D008190:Lyases; D006836:Hydro-Lyases; C082741:scytalone dehydratase; C071445:linA protein, Pseudomonas paucimobilis; D013251:Steroid Isomerases; C022660:steroid delta-isomerase