Catechol 2,3-dioxygenases (C23Os; EC 184.108.40.206) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylEP (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylES (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylES sequence, except that its C-terminal end was derived from XylEP. While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylEP sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylEP and XylES. According to the crystal structure of XylEP, the C-terminal region is involved in the formation of a quaternary structure. Amino acid differences between HB3-C23O and HB4-C23O included the specific β-strand for oligomerization. Thus the quaternary structures of active C23Os, XylES, XylEP and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylES, XylEP and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer. We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.
Abbreviations used: C23O, catechol 2,3-dioxygenase; IPTG, isopropyl β-d-thiogalactoside.
Present address: Japan Biological Information Research Center, JBIC, 2-41-6 Aomi, Kouto-ku, Tokyo 135-0064, Japan.
Present address: Research Institute of Molecular Genetics, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan.
The nucleotide sequence data reported will appear in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AB074513 and AB074514.