Discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in Hydrogenobacter thermophilus

Abstract

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent phosphoglycerate mutase (dPGM). However, because they possessed neither mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward l-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from "dPGM-like" proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism.

FIGURE 1.

General serine anabolic pathways. PGM, phosphoglycerate mutase; THF, tetrahydrofolate.

FIGURE 2.

12% SDS-PAGE analysis of purified native iPSPs (A), heterologously expressed H. thermophilus PSPs, and an iPSP homolog from T. thermophilus (B). A, lane 1, molecular mass markers; lane 2, 0.2 μg of purified protein I (htPSP1); lane 3, 0.5 μg of purified protein II (htPSP2). The arrowheads indicate the bands corresponding to proteins I and II. B, heterologously expressed and purified proteins. Lane 1, 2 μg of purified protein I; lane 2, 2 μg of purified protein II; lane 3, 2 μg of TTHA0368-coded protein; lane 4, molecular mass markers.