Tài liệu Báo cáo khoa học: Effect of ionic strength and oxidation on the P-loop conformation of the

Effect of ionic strength and oxidation on the P-loop

conformation of the protein tyrosine phosphatase-like

phytase, PhyAsr

Robert J. Gruninger1

, L. Brent Selinger2 and Steven C. Mosimann1

1 Department of Chemistry and Biochemistry, University of Lethbridge, Canada

2 Department of Biological Sciences, University of Lethbridge, Canada

Enzymes that degrade myo-inositol-1,2,3,4,5,6-

hexakisphosphate (InsP6) are ubiquitous in nature and

have been identified in prokaryotes, protists, fungi,

animals, and plants [1,2]. InsP6 is the most abundant

inositol phosphate in the cell, and has been implicated

in important cellular processes, including DNA repair,

mRNA export, RNA editing, cellular signaling, endo￾cytosis, and vesicular trafficking [3–6]. The generic

term phytase is applied to enzymes that hydrolyze

InsP6 into inorganic phosphate and various lower

phosphorylated myo-inositols. The recently described

protein tyrosine phosphatase (PTP)-like phytase from

Selenomonas ruminantium, PhyAsr, contains the PTP

active site signature sequence (HCX5RS ⁄T), is structur￾ally similar to PTPs, and utilizes the same catalytic

mechanism as PTPs to hydrolyze phosphodiester

bonds [7,8]. Although the biological function of

these PTP-like phytases is unclear, they are the first

Keywords

ionic strength; oxidation; phytase; P-loop;

protein tyrosine phosphatase

Correspondence

S. C. Mosimann, Department of Chemistry

and Biochemistry, University of Lethbridge,

Lethbridge, AB, Canada T1K 3M4

Fax: +1 403 329 2057

Tel: +1 403 329 2283

E-mail: [email protected]

Database

The coordinates and structure factors for

the structures of PhyAsr at ionic strengths

of 200, 300, 400 and 500 mM and with the

catalytic cysteine oxidized to cysteine

sulfonic acid have been deposited in the

Protein Data Bank (entries 2PSZ, 3D1O,

3D1Q, 3D1H and 2PT0, respectively)

(Received 26 March 2008, revised 21 May

2008, accepted 27 May 2008)

doi:10.1111/j.1742-4658.2008.06524.x

The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Seleno￾monas ruminantium is a novel member of the PTP superfamily, and the

only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-

hexakisphosphate. In addition to the unique substrate specificity of PhyAsr,

the phosphate-binding loop (P-loop) has been reported to undergo a con￾formational change from an open (inactive) to a closed (active) conforma￾tion upon ligand binding at low ionic strength. At high ionic strengths, the

P-loop was observed in the closed, active conformation in both the pres￾ence and absence of ligand. To test whether the P-loop movement can be

induced by changes in ionic strength, we examined the effect that ionic

strength has on the catalytic efficiency of PhyAsr, and determined the

structure of the enzyme at several ionic strengths. The catalytic efficiency

of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in

kcat ⁄ Km and a ninefold decrease in Km when the ionic strength is increased

from 100 to 500 mm. Surprisingly, the P-loop is observed in the catalyti￾cally competent conformation at all ionic strengths, despite the absence of

a ligand. Here we provide structural evidence that the ionic strength depen￾dence of PhyAsr and the conformational change in the P-loop are not

linked. Furthermore, we demonstrate that the previously reported P-loop

conformational change is a result of irreversible oxidation of the active site

thiolate. Finally, we rationalize the observed P-loop conformational

changes observed in all oxidized PTP structures.

Abbreviations

Cdc25B, cell division cycle 25 homolog B; InsP6, myo-inositol hexakisphosphate; PhyAsr, Selenomonas ruminantium protein tyrosine

phosphatase-like phytase; P-loop, phosphate-binding loop; PTP, protein tyrosine phosphatase; RPTPa, receptor protein tyrosine phosphatase

alpha; Yop51, Yersinia protein tyrosine phosphatase.

FEBS Journal 275 (2008) 3783–3792 ª 2008 The Authors Journal compilation ª 2008 FEBS 3783