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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.

SUBMITTER: Liu S

PROVIDER: S-EPMC1196051 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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

Liu Shenghao S Ogawa Naoto N Senda Toshiya T Hasebe Akira A Miyashita Kiyotaka K

Journal of bacteriology 20050801 15

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 ...[more]

PMID: 16030237

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Project description:The constant increase in the number of solved protein structures is of great help in understanding the basic principles behind protein folding and evolution. 3-D structural knowledge is valuable in designing and developing methods for comparison, modelling and prediction of protein structures. These approaches for structure analysis can be directly implicated in studying protein function and for drug design. The backbone of a protein structure favours certain local conformations which include ?-helices, ?-strands and turns. Libraries of limited number of local conformations (Structural Alphabets) were developed in the past to obtain a useful categorization of backbone conformation. Protein Block (PB) is one such Structural Alphabet that gave a reasonable structure approximation of 0.42 Å. In this study, we use PB description of local structures to analyse conformations that are preferred sites for structural variations and insertions, among group of related folds. This knowledge can be utilized in improving tools for structure comparison that work by analysing local structure similarities. Conformational differences between homologous proteins are known to occur often in the regions comprising turns and loops. Interestingly, these differences are found to have specific preferences depending upon the structural classes of proteins. Such class-specific preferences are mainly seen in the all-? class with changes involving short helical conformations and hairpin turns. A test carried out on a benchmark dataset also indicates that the use of knowledge on the class specific variations can improve the performance of a PB based structure comparison approach. The preference for the indel sites also seem to be confined to a few backbone conformations involving ?-turns and helix C-caps. These are mainly associated with short loops joining the regular secondary structures that mediate a reversal in the chain direction. Rare ?-turns of type I' and II' are also identified as preferred sites for insertions.

Dataset Information

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

Publications

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

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