The plasmid-encoded enzymes characterized to date differ from their chromosomally encoded counterparts as e.g. the three MDH enzymes exhibit different biochemical and physical properties and their genes are regulated differently [23]. GlpXC was shown to be the major FBPase of B. methanolicus, while GlpXP also carries SBPase activity [28]. Both FBAC and FBAP AZD5363 mw are SBAs, but their kinetic parameters allowed to

distinguish FBAC as major glycolytic FBA and FBAP as major gluconeogenic FBA [26]. The objective of this study was to characterize the role and enzymatic properties of the two TKTs from B. methanolicus to get further insight into the genetic and biochemical aspects of methylotrophy Results Bioinformatic analysis and phylogeny of the TKTP and TKTC from B. methanolicus B. methanolicus possesses two distinct genes encoding TKT [21], tkt C on the chromosome and the plasmid located tkt P . The deduced primary sequences of these proteins show a similarity of 87% MI-503 mouse (578/668) and an identity of 76% (506/668) to each other. The closest homolog of

TKTC present in the database is the chromosomally encoded homolog (EIJ77615.1; 97% identical amino acids) of B. methanolicus strain PB1. Similarly, the closest homolog of plasmid encoded TKTP is the TKT (EIJ81398.1) from B. methanolicus PB1 (95% identical amino acids), which is encoded on plasmid pBM20. BLAST analyses of the amino acid sequences of TKTC and TKTP as queries suggested their classification as TKT with more than 200 sequences sharing 50% or more identical amino acids. An amino acid sequence alignment

with biochemically characterized and experimentally verified TKTs from E. coli K12, encoded by tktA and tktB[12, 30, 31], Plasmodium falciparum, encoded by pftk[32], Leishmania mexicana, encoded by tkt[33], Trypanosoma brucei, encoded by tbtkt[34], and Saccharomyces cerevisiae, encoded by sctk[35] revealed the presence of highly conserved amino acid residues throughout the sequence with Histamine H2 receptor two notable motifs (Figure 2). The first N- terminal located motif is common to all Thiamindiphosphat (ThDP)-dependent enzymes. The sequence begins with the highly conserved residues Gly-Asp-Gly (GDG) followed by 21 less conserved residues [36, 37]. The second click here so-called Tk motif is specific for all TKTs [38]. Figure 2 Primary sequence alignment of TKT proteins. Black and grey boxes indicate identical and similar residues. Bars indicate the characteristic ThDP motif and the TK motif. The sequence alignment was carried out using ClustalW, the alignment was formatted using BoxShade. Overexpression of tkt C and tkt P resulted in increased TKT activity in B. methanolicus In order to study if the tkt C and tkt P genes encode functionally active TKT enzymes, both genes were overexpressed in B. methanolicus.