Tài liệu Báo cáo khoa học: X-ray crystallographic and NMR studies of pantothenate synthetase provide

X-ray crystallographic and NMR studies of pantothenate

synthetase provide insights into the mechanism of

homotropic inhibition by pantoate

Kalyan Sundar Chakrabarti*, Krishan Gopal Thakur, B. Gopal and Siddhartha P. Sarma

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Keywords

competitive inhibition; NMR; pantothenate

biosynthesis; substrate binding; X-ray

crystallography

Correspondence

S. P. Sarma, 207, Molecular Biophysics

Unit, Indian Institute of Science, Bangalore

560012, India

Fax: +91 80 23600535

Tel: +91 80 22932839

E-mail: [email protected]

*Present address

Department of Biochemistry and Howard

Hughes Medical Institute, MS009 Brandeis

University, Waltham, MA, USA

Database

The 1

HN, 15N, 13Ca

, 13Cb and 13C¢ chemical

shift values of the dimeric N-terminal

domain of Escherichia coli pantothenate

synthetase have been deposited in

BioMagResBank (http://www.bmrb.

wisc.edu) under the accession number 6940

The structure factor file and the atomic

coordinates of the dimeric N-terminal

domain of Escherichia coli pantothenate

synthetase bound to three molecules of

pantoate have been deposited in the Protein

Data Bank under the accession number

3GUZ

(Received 26 July 2009, revised 6 October

2009, accepted 24 November 2009)

doi:10.1111/j.1742-4658.2009.07515.x

The structural basis for the homotropic inhibition of pantothenate synthe￾tase by the substrate pantoate was investigated by X-ray crystallography

and high-resolution NMR spectroscopic methods. The tertiary structure of

the dimeric N-terminal domain of Escherichia coli pantothenate synthetase,

determined by X-ray crystallography to a resolution of 1.7 A˚ , showed a

second molecule of pantoate bound in the ATP-binding pocket. Pantoate

binding to the ATP-binding site induced large changes in structure, mainly

for backbone and side chain atoms of residues in the ATP binding

HXGH(34–37) motif. Sequence-specific NMR resonance assignments and

solution secondary structure of the dimeric N-terminal domain, obtained

using samples enriched in 2

H, 13C, and 15N, indicated that the secondary

structural elements were conserved in solution. Nitrogen-15 edited two￾dimensional solution NMR chemical shift mapping experiments revealed

that pantoate, at 10 mm, bound at these two independent sites. The solu￾tion NMR studies unambiguously demonstrated that ATP stoichiometri￾cally displaced pantoate from the ATP-binding site. All NMR and X-ray

studies were conducted at substrate concentrations used for enzymatic

characterization of pantothenate synthetase from different sources [Jonczyk

R & Genschel U (2006) J Biol Chem 281, 37435–37446]. As pantoate bind￾ing to its canonical site is structurally conserved, these results demonstrate

that the observed homotropic effects of pantoate on pantothenate biosyn￾thesis are caused by competitive binding of this substrate to the ATP-bind￾ing site. The results presented here have implications for the design and

development of potential antibacterial and herbicidal agents.

Structured digital abstract

l MINT-7301221: PS (uniprotkb:P31663) and PS (uniprotkb:P31663) bind (MI:0407) by x-ray

crystallography (MI:0114)

l MINT-7301241: PS (uniprotkb:P31663) and PS (uniprotkb:P31663) bind (MI:0407) by

nuclear magnetic resonance (MI:0077)

Abbreviations

cPS, C-terminal domain of pantothenate synthetase; HSQC, heteronuclear single quantum coherence; nPS, dimeric N-terminal domain of

Escherichia coli pantothenate synthetase; PS, pantothenate synthetase; TEV, tobacco etch virus; TROSY, transverse relaxation optimized

spectroscopy.

FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 697