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Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

ABSTRACT: Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.

SUBMITTER: Kim SI

PROVIDER: S-EPMC179385 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Kim S I SI Leem S H SH Choi J S JS Chung Y H YH Kim S S Park Y M YM Park Y K YK Lee Y N YN Ha K S KS

Journal of bacteriology 19970801 16

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR ...[more]

PMID: 9260969

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Project description:BACKGROUND: Argonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing. Various classes of Argonaute genes have been identified from plants and might be involved in developmental regulation. However, little is known about these genes in wheat (Triticum aestivum). RESULTS: In this study, two full-length cDNAs of Argonaute were cloned from wheat, designated as TaAGO1b and TaAGO4. The cDNA of TaAGO1b is 3273 bp long and encodes 868 amino acids, with a predicted molecular weight of ~97.78 kDa and pI of 9.29. The 3157-bp TaAGO4 encodes 916 amino acids, with a molecular mass of 102.10 kDa and pI of 9.12. Genomics analysis showed that TaAGO1b and TaAGO4 contain 20 and 18 introns, respectively. Protein structural analysis demonstrated that typical PAZ and PIWI domains were found in both TaAGO1b and TaAGO4. From the highly conserved PIWI domains, we detected conserved Asp-Asp-His (DDH) motifs that function as a catalytic triad and have critical roles during the process of sequence-specific cleavage in the RNAi machinery. Structural modelling indicated that both TaAGOs can fold to a specific ?/? structure. Moreover, the three aligned DDH residues are spatially close to each other at the "slicer" site of the PIWI domain. Expression analysis indicated that both genes are ubiquitously expressed in vegetative and reproductive organs, including the root, stem, leaf, anther, ovule, and seed. However, they are differentially expressed in germinating endosperm tissues. We were interested to learn that the two TaAGOs are also differentially expressed in developing wheat plants and that their expression patterns are variously affected by vernalization treatment. Further investigation revealed that they can be induced by cold accumulation during vernalization. CONCLUSIONS: Two putative wheat Argonaute genes, TaAGO1b and TaAGO4, were cloned. Phylogenetic analysis, prediction of conserved domains and catalytic motifs, and modelling of their protein structures suggested that they encode functional Argonaute proteins. Temporal and spatial expression analyses indicated that these genes are potentially involved in developmental regulation of wheat plants.

Dataset Information

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Publications

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

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