Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase

Abstract

We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E. coli and performed functional studies of the recombinant enzymes. Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E. histolytica genes, such as transcripts with relatively short 5′ and 3′ untranslated flanking regions containing the conserved E. histolytica transcription promoter elements located 5′ to the initiation codon and a polyadenylation signal in the 3′ UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts. At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29 amino acid LID region present in the long variant isoenzymes. EhAK was 30–38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48–49% identity) to UMP/CMP kinases. Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP. Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes.