Amino acids in positions 48, 52, and 73 differentiate the substrate specificities of the highly homologous chlorocatechol 1,2-dioxygenases CbnA and TcbC

Abstract

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.

FIG. 1.

Alignment of (chloro)catechol 1,2-dioxygenases. Unless stated otherwise below, the amino acid sequences used for the alignment are those of chlorocatechol 1,2-dioxygenase: CatA ADP1, catechol 1,2-dioxygenase from Acinetobacter sp. strain ADP1 (DDBJ accession number AF009224); CbnA NH9, Ralstonia eutropha NH9 (accession number AB019032); TcbC P51, Pseudomonas sp. strain P51 (accession number M57629); TfdC CSV90, 3,5-dichlorocatechol 1,2-dioxygenase from Alcaligenes sp. CSV90 (accession number D16356), identical to TfdC of R. eutropha JMP134 (3, 41); TfdC NK8, Burkholderia sp. NK8 (accession number AB050198); and ClcA 1CP, 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP (accession number AF003948). The alignment is according to the study of ClcA of R. opacus 1CP (17). The conserved residues in all six enzymes are shown in boldface. The 12 residues differing between CbnA and TcbC are expressed in boldface italics, and numbers of the positions are indicated in italics above the alignment. The four regions delineated by restriction sites in cbnA and tcbC are indicated by the dotted arrows above the alignment. The residues which are considered to interact with substrate in CatA of ADP1 and ClcA of 1CP are labeled with # above the alignment and with * below the alignment, respectively. The four conserved ligands for iron are labeled with δ.

FIG. 2.

Molecular modeling of CbnA and TcbC with substrates. (a) CbnA and 3,5-dichlorocatechol; (b) TcbC and 3,4-dichlorocatechol. Molecular models of CbnA and TcbC were prepared on the basis of the crystal structures of ClcA from Rhodococcus opacus 1CP (Protein Database identification no. 1s9a) (17) and CatA from Acinetobacter sp. ADP1 (Protein Database identification no. 1dlm and 1dlq) (65).