Marie Wintzer (Riken)| Ryuhei Nakamura (Riken, ELSI, Tokyo Institute of Technology)| Yoko Chiba (Riken)
Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine, and is found in all three domains of life. Classic, metal-dependent PSPs (dPSPs) belong to the haloacid dehalogenase-like hydrolase superfamily. Some organisms, however, lack a known PSP even though they exhibit a functional phosphoserine synthetic pathway, and should then possess a genetically new type of PSP. Hydrogenobacter thermophilus, a thermophilic, obligate chemolithoautotroph in the phylum Aquificae, possesses two novel-type, metal-independent PSP (iPSP). These proteins, named HtPspA and HtPspB, belong to the histidine phosphatase superfamily. Despite their high primary structure similarity and the conservation of residues essential for phosphatase activity, HtPspA and HtPspB present notable differences. In H. thermophilus they are expressed as homodimers (A-A) or heterodimers (A-B). However, only HtPspA shows significant PSP activity, and is therefore the catalytic subunit as a PSP. HtPspB does not have substantial PSP activity and is only soluble in the presence of HtPspA, at least when it is expressed in E. coli. Crystal structure analysis revealed that His85 and the C-terminal region, which exist in HtPspA but not in HtPspB, are necessary for PSP activity in this organism.
Thermosulfidibacter takaii, which belongs to a distinct family in the phylum Aquificae, possesses only one homolog of iPSP which lacks His85 and the C-terminal region, showing higher identity to HtPspB than to HtPspA. To examine whether this homolog can serve as a serine synthetic enzyme in T. takaii - which should have a serine synthetic pathway - while lacking the structural units of HtPspA essential for PSP activity, we characterized its enzymatic properties. In contrast to HtPspB, TtPsp was expressed in the soluble fraction in E. coli. It is thermostable at 70°C, the optimal growth temperature of T. takaii, and is likely to form a homodimer as suggested by gel chromatography. Enzymatic activity assays revealed that TtPsp possesses only about 1% of the PSP activity (per mole of protein) exhibited by HtPspA, and displays higher activity towards L-phosphothreonine than L-phosphoserine. The phylogenetic characterization of TtPspB showed that this protein forms a clade with PspB homologs of distinct families within the phylum Aquificae.
Our study revealed that an ancestor of Aquificae already possessed PspA and PspB, and that the lack of a PspA homolog in T. takaii was caused by gene loss. Whether the common ancestor of PspA and PspB was a functional PSP or not, and the physiological function of PspB, is still unknown.