Molecular and biochemical analysis of the β class carbonic anhydrases in Caenorhabditis elegans

Abstract

The beta class of the carbonic anhydrase (CA) enzyme family has been found in plants, yeast, bacteria and algae, but not in animals. Also, little is known concerning the CAs of C. elegans. Genes possibly encoding beta-CAs were revealed by in silico analysis of the C. elegans genome. Amino acid sequence and 3D structure analysis revealed a resemblance to both plant and cab-type beta-CAs. Temporal expression patterns of the two genes, as well as changes in expression levels under different atmospheric conditions (stress) were analyzed by real-time RT-PCR. Recombinant enzymes, expressed in E. coli were used for in vitro measurement of CA activity, while a yeast complementation experiment was performed in order to assess their ability to complement the function of S. crevisieae beta-CA (NCE103) in vivo. RNAi by feeding was performed on wild-type populations that were then examined for a visible phenotype under normal or various stress conditions (pH, CO(2)/O(2)). Two genes possibly encoding beta-CAs were revealed (bca-1 and y116a8c.28). Their products contain elements of both plant and cab-type CAs. Both assays showed that Y116a8c.28 is an active CA. Both genes showed significant levels of transcript accumulation during development, while they also responded to the stress conditions. No visible phenotype was scored under normal or stress conditions.

Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

“…Fasseas et al [37] state that the 3D structure of their C. elegans β-CA models would resemble archaeal "cab-type" β-CAs, but we cannot agree with their conclusion. In our eyes, the models are nearly identical to the pea BCA (panel a in their Figure 2), and in our hands SwissModel http://swissmodel.expasy.org/ in fact chooses the pea β-CA structure as a template for both insect and nematode β-CAs.…”

“…The exception is nematodes, which seem to have two β -CA genes in their genomes. A very recent article [37] reported the cloning and characterization of C. elegans β-CAs and the authors found that one of the two isoforms, BCA-1, has no activity and does not work in complementation assay. This might, however, be due to incorrect sequence: the sequence they show for C. elegans BCA-1 contains the coding sequence of the preceding MTP18 gene fused to the β -CA reading frame.…”

Bioinformatic analysis of beta carbonic anhydrase sequences from protozoans and metazoans

“…This class of enzyme has also been discovered in a wide range of taxa, such as yeast ( Saccharomyces cerevisiae ) [34-36], cyanobacteria ( Synechocystis sp. PCC6803) [37], carboxysomes of chemoautotrophic bacteria ( Halothiobacillus neapolitanus ) [38], green algae ( Chlamydomonas reinhardtii ) [39], red algae ( Porphyridium purpureum ) [40], nematodes ( Caenorhabditis elegans) [41], and insects ( Drosophila melanogaster) [4]. While β-CAs were initially thought to be expressed only in plants, this enzyme family is indeed present in a wide variety of species – from bacteria and archaea to invertebrate animals, missing only from vertebrates and most chordates, making it an attractive target for evolutionary studies [5].…”

“…The first metazoan β-CAs were reported in 2010 [41]. In one of the studies [4,41], two genes encoding β-CAs (y116a8c.28 and bca-1) were identified in Caenorhabditis elegans .…”

“…In one of the studies [4,41], two genes encoding β-CAs (y116a8c.28 and bca-1) were identified in Caenorhabditis elegans . Another study reported a novel β-CA gene identified from FlyBase, which was named DmBCA (short for Drosophila melanogaster β-CA) [4].…”