Molecular Characterization of the C-Terminus Kinase Containing Domain of a Triphosphate Tunnel Metallo-enzyme Family Protein (AtTTM2) from Arabidopsis thaliana

Abstract

Three loci, At1g73980, At1g26190 and At2g11890, are annotated as the CYTH domains in the Arabidopsis genome and coding for the AtTTM1, AtTMM2 and AtTTM3 proteins respectively. These proteins belong to the triphosphate tunnel metalloenzymes (TTM) family and members of this extended family act on triphosphate substrates and require a divalent cationic cofactor, usually Mg²⁺and/or Mn²⁺. They are therefore referred to as TTMs. TTMs are known as a group of enzymes that can hydrolyse a range of tripolyphosphates. Promoter swap analysis has shown that the AtTTM1-2 can functionally complement each other. Moreover, all the TTMs display the same biochemical properties but distinct biological functions governed by their transcriptional regulation. Based on the fact that AtTTM2 has recently been annotated as a possible kinase, this work then primarily focused on the functional characterisation of its possible kinase activity in the Arabidopsis thaliana plant and possibly other related higher plants. The work involved germinating some A. thaliana seeds, followed by extraction of total RNA from the generated plants. Amplification of the targeted and desired AtTTM2 gene fragment was performed using a RT-PCR system. The amplified AtTTM2 coding region was ligated into a TrHis2-TOPO expression vector in preparation for recombinant protein expression. The ligated AtTTM2 coding region was then used to transform competent E. coli BL21 (DE3) pLysS cells followed by partial expression of the recombinant AtTTM2 protein. Using a crude extract of the recombinant protein, the predicted kinase activity of the AtTTM2 protein confirmed using the OmniaTM Recombinant system. After this, the protein was further assessed and analysed bioinformatically to determine its possible physiological and functional roles in Arabidopsis and other related higher plants. The generated information showed that the AtTTM2 protein is indeed a bona fide plant kinase that is highly expressed in various plant tissues during the different stages of plant growth and development.