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Identification of protein fold and catalytic residues of γ‐hexachlorocyclohexane dehydrochlorinase LinA
Author(s) -
Nagata Yuji,
Mori Katsuki,
Takagi Masamichi,
Murzin Alexey G.,
Damborský Jiří
Publication year - 2001
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10007
Subject(s) - site directed mutagenesis , homology modeling , mutagenesis , biochemistry , mutant , biology , peptide sequence , chemistry , sequence alignment , homology (biology) , active site , stereochemistry , amino acid , enzyme , gene
γ‐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‐Δ 5 ‐steroid isomerase, nuclear transport factor 2, and the β‐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. Proteins 2001;45:471–477. © 2001 Wiley‐Liss, Inc.