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

Homoserine Phosphotransferase Activity

S. Kumar Singh, Kun Yang, Subramanian Karthikeyan, Tu Huynh, Xuejun Zhang, Margaret A. Phillips, Hong Zhang

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 apo-form 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. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a Km of 0.207 mM and kcat 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.

Original languageEnglish (US)
Pages (from-to)13166-13173
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number13
DOIs
StatePublished - Mar 26 2004

Fingerprint

Homoserine
Phosphoserine
Enzyme activity
Pseudomonas aeruginosa
Phosphotransferases
Genes
homoserine kinase
Assays
Enzymes
High pressure liquid chromatography
Spectrophotometry
Crystal structure
Phosphates
High Pressure Liquid Chromatography
phosphoserine phosphatase
Primary Spontaneous Pneumothorax
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

The thrH Gene Product of Pseudomonas aeruginosa Is a Dual Activity Enzyme with a Novel Phosphoserine : Homoserine Phosphotransferase Activity. / Singh, S. Kumar; Yang, Kun; Karthikeyan, Subramanian; Huynh, Tu; Zhang, Xuejun; Phillips, Margaret A.; Zhang, Hong.

In: Journal of Biological Chemistry, Vol. 279, No. 13, 26.03.2004, p. 13166-13173.

Research output: Contribution to journalArticle

@article{859697cf2516463c86c3a906cb5a1ec5,
title = "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 apo-form 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. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a Km of 0.207 mM and kcat 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.",
author = "Singh, {S. Kumar} and Kun Yang and Subramanian Karthikeyan and Tu Huynh and Xuejun Zhang and Phillips, {Margaret A.} and Hong Zhang",
year = "2004",
month = "3",
day = "26",
doi = "10.1074/jbc.M311393200",
language = "English (US)",
volume = "279",
pages = "13166--13173",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "13",

}

TY - JOUR

T1 - The thrH Gene Product of Pseudomonas aeruginosa Is a Dual Activity Enzyme with a Novel Phosphoserine

T2 - Homoserine Phosphotransferase Activity

AU - Singh, S. Kumar

AU - Yang, Kun

AU - Karthikeyan, Subramanian

AU - Huynh, Tu

AU - Zhang, Xuejun

AU - Phillips, Margaret A.

AU - Zhang, Hong

PY - 2004/3/26

Y1 - 2004/3/26

N2 - 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 apo-form 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. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a Km of 0.207 mM and kcat 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.

AB - 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 apo-form 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. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a Km of 0.207 mM and kcat 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.

UR - http://www.scopus.com/inward/record.url?scp=1842424658&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842424658&partnerID=8YFLogxK

U2 - 10.1074/jbc.M311393200

DO - 10.1074/jbc.M311393200

M3 - Article

VL - 279

SP - 13166

EP - 13173

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 13

ER -