Abstract
Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) 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 XylE(P) (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylE(S) (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylE(S) sequence, except that its C-terminal end was derived from XylE(P). While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylE(P) 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 XylE(P) and XylE(S). According to the crystal structure of XylE(P), 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 beta-strand for oligomerization. Thus the quaternary structures of active C23Os, XylE(S), XylE(P) and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylE(S), XylE(P) 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.