The thrH Gene Product of Pseudomonas aeruginosa Is a Dual Activity Enzyme with a Novel Phosphoserine:Homoserine Phosphotransferase Activity

Abstract

The thrH gene product of Pseudomonas aeruginosa has been shown to complement both homoserine kinase (thrB gene product) and phosphoserine phosphatase (serB gene product) activities in vivo. Sequence comparison has revealed that ThrH is related to phosphoserine phosphatases (PSP, EC 3.1.3.3) and belongs to the L-2-haloacid dehalogenase-like protein superfamily. We have solved the crystal structures of ThrH in the apoform and in complex with a bound product phosphate. The structure confirms an overall fold similar to that of PSP. Most of the catalytic residues of PSP are also conserved in ThrH, suggesting that similar catalytic mechanisms are used by both enzymes. Spectrophotometrybased in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a K m of 0.207 mM and k cat of 13.4 min ؊1 , comparable with those of other PSPs. More interestingly, using high pressure liquid chromatography-based assays, we have demonstrated that ThrH is able to further transfer the phosphoryl group to homoserine using phosphoserine as the phosphoryl group donor, indicating that ThrH has a novel phosphoserine:homoserine phosphotransferase activity.In most bacteria, a major portion of endogenous threonine is generated from homoserine (Hse) 1 in a two-step pathway: 1) phosphorylation of homoserine to phosphohomoserine (P-Hse), a reaction catalyzed by homoserine kinase (thrB gene product), followed by 2) isomerization and dephosphorylation of P-Hse to produce L-threonine (1). On the other hand, serine is synthesized from phosphoglycerate through sequential reactions catalyzed by 3-phosphoglycerate dehydrogenase (SerA), 3-phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB) (2). ThrB and SerB thus function in two distinct pathways of amino acid biosynthesis. In Escherichia coli and many other species, inactivation of thrB gene results in threonine auxotrophy (3). However, in Pseudomonas aeruginosa, thrB mutants do not lead to threonine auxotrophy (4). A new gene named thrH has been identified that complements homoserine kinase activity in vivo (5). Furthermore, overexpression of ThrH also rescues serine auxotrophy caused by serB mutant in both P. aeruginosa and E. coli (5). In vitro assays have confirmed the phosphoserine phosphotase (PSP) activity of ThrH, but no homoserine kinase (HSK) activity of ThrH has been detected by the standard coupled kinase assays (5).Sequence comparison reveals that ThrH protein is homologous to the PSP encoded by serB gene, which belongs to the HAD (L-2-haloacid dehalogenase)-like hydrolase superfamily (5, 6). Several members of this superfamily including phosphoserine phosphatase, phosphoglycolate phosphatase, phosphomannomutase, and ␤-phosphoglucomutase use a aspartylphosphate phosphoenzyme intermediate during catalysis (7,8). Recent structural studies on these enzymes in complex with a series of substrates and transition state intermediates provided a wealth of structural data delineating the catalytic mechanism of this class of aspartyl-utilyzing enzymes (9 -12). ThrH...

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

“…These kinetic constants were observed to be lower in comparison with those obtained for PSP enzymes characterized from either Hydrogenobacter thermophiles (K m of 1.6 mM), P. gingivalis (K m of 2.0 mM and 2.6 mM for phosphoserine peptides), or Pseudomonas aeruginosa (K m of 207 M) (26,36,37). We also observed that maximal SerB2 activity was observed in the initial 5 min and that inclusion of 0.01% Triton X-100 enhanced SerB2 activity by 15-20% (Fig.…”

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

“…Specific roles in amino acid metabolism continued to be acquired in specific lineages of the bacterial tree; for example, the enolase phosphatase and the phosphoserine:homoserine phosphotransferase respectively in methionine and threonine metabolism. 139,140,150 The other major bacterial innovations were related to sugar metabolism and appear to have occurred somewhat later in bacterial evolution. These sugar metabolism enzymes arose throughout the HAD superfamily, though the cof assemblage appears to be the most dominant amongst them.…”

“…The related ThrH subfamily, which is restricted to the proteobacteria, participates in the threonine biosynthesis pathway by catalyzing a phosphoserine-homoserine phosphotransfer reaction, similar to the phosphate exchange reaction of SerB. 149,150 The PHOSPHO1 subfamily contains a peculiar C2 cap, which has three conserved cysteine residues, suggesting that it is stabilized by metal chelation. The vertebrate versions of this subfamily are believed to mobilize inorganic phosphate for skeletal matrix mineralization mineralization through their action on phosphocholine and phosphoethanolamine.…”

Characterization of a New Pantothenate Kinase Isoform from Helicobacter pylori

“…These included GTP, CTP, UTP, PEP, and acetyl-and carbamoylphosphate. Phosphoserine and phosphothreonine were also tested, since they are known substrates of some phosphotransferase enzymes (21,22). The reaction mixture with PEP was subsequently analyzed by the two enzymecoupled assay, while the rest of the reaction mixtures were analyzed by ESI-MS for the presence of 4Ј-phosphopantothenate.…”