brasilense Sp7. Results Sequence and phylogenetic analysis of gca1 of A.
brasilense A search for the presence of ORFs annotated as carbonic anhydrase in the genome of A. brasilense Sp245 http://genome.ornl.gov/microbial/abra/ revealed three ORFs out of which two were annotated to encode carbonic anhydrase/acetyltransferase. BLAST results of the amino acid sequences of these two ORFs showed homology with putative γ-CAs. Using the sequence information from A. brasilense Sp245 genome, one of the putative γ-CA ORF (gca1) of A. brasilense Sp7 was PCR amplified, and sequenced. The nucleotide and deduced amino acid sequence VX-770 price of the A. brasilense Sp7 gca1 and the putative γ-CA of A. brasilense Sp245 were 97% and 99% identical, respectively. The gca1 ORF consisted of 519 bp, which can translate a polypeptide of 173 amino acids with a predicted molecular mass of 19 kDa. BLASTP analysis of the deduced amino acid sequence of A. brasilense Gca1 revealed 27% identity with Cam, a γ-CA from M. thermophila. In addition to its homology with putative γ-CAs, Gca1 also showed significant homology to proteins annotated as acetyltransferase/isoleucine patch superfamily with no
predicted function (unknown proteins). As inferred from X-ray crystallographic studies of Cam, the active-site zinc is coordinated by three histidine residues [9]. The alignment of Gca1 with the Cam sequence showed that the essential histidines (His-81, His-117 and His-122) required for ligating the active site Zn are absolutely conserved in Gca1. Further analysis revealed that three SP600125 datasheet other residues (Arg-59, Asp-61 and Gln-75) present in all γ-class CA sequences and reported to be involved in biochemical activity of Cam of M. thermophila, are also conserved in Gca1 (Additional
file 1 Figure S1). Two glutamate residues, Glu-62 and find more Glu-84 of Cam, whose role has been shown in CO2 hydration and proton transfer, respectively, are conserved in cyanobacterial CcmM sequence but neither in Gca1 nor in other γ-CA homologues such as Pseudomonas putida (PhaM) and E. coli (CaiE) which share 36%, and 32% identity, respectively, with Gca1, suggesting that alternative residues might serve these roles. To examine the phylogenetic relationship of A. brasilense cAMP Gca1 with other known orthologs, the amino acid sequences of different γ-CAs from eukaryotic photosynthetic organisms, cyanobacteria, bacteria and archaea were used to generate multiple sequence alignment and a phylogenetic tree (Figure 1). The deduced γ-CA amino acid sequences clustered in two clades; the larger Clade A consisted of sequences from all three domains of life. The catalytically important residues of Cam, Glu-62 and Glu-84 were missing in these sequences and information regarding CA activity of protein encoded by any of these sequences is lacking. Clade B consisted of well documented Cam protein from M. thermophila and cyanobacterial CcmM proteins.