Identification of protein fold and catalytic residues of gamma-hexachlorocyclohexane dehydrochlorinase LinA

gamma -Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydroch lorinase that has no homologous sequence at the amino acid-sequence level a nd for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of proteins containing seytal one dehydratase, 3-oxo-Delta (5)-steroid isomerase, nuclear transport facto r 2, and the P-subunit of naphthalene dioxygenase-all known structures with different functions. The catalytic and the active site residues of LinA ar e predicted on the basis of its homology model. Nine mutants that carry sub stitutions of the proposed catalytic residues were constructed by site-dire cted mutagenesis. In addition to these, eight mutants that have a potential to make contact with the substrate were prepared by site-directed mutagene sis. 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 v alidity. We conclude that Lin-A catalyzes the proton abstraction via the ca talytic dyad H73-D25 by a similar mechanism as described for scytalone dehy dratase. The results suggest that LinA and scytalone dehydratase evolved fr om a common ancestor. LinA may have evolved from an enzyme showing a dehydr atase activity. (C) 2001 Wiley-Liss, Inc.