Tài liệu Báo cáo khoa học: Crystal structures of a bacterial 6-phosphogluconate dehydrogenase reveal

Crystal structures of a bacterial 6-phosphogluconate

dehydrogenase reveal aspects of specificity, mechanism

and mode of inhibition by analogues of high-energy

reaction intermediates

Ramasubramanian Sundaramoorthy1

, Jorge Iulek1,2, Michael P. Barrett3

, Olivier Bidet4

,

Gian Filippo Ruda1

, Ian H. Gilbert1 and William N. Hunter1

1 Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences, University of Dundee, UK

2 Department of Chemistry, Biotechnology Center, State University of Ponta Grossa, Parana´, Brazil

3 Division of Infection & Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, UK

4 Welsh School of Pharmacy, Cardiff University, UK

The pentose phosphate pathway is an anabolic path￾way, the major functions of which are production

of ribose 5-phosphate, utilized in the biosynthesis of

nucleotides, and to maintain a pool of NADPH [1].

The NADPH serves to alleviate the oxidative stress of

aerobic metabolism and participates in varied bio￾synthetic processes [2]. The third enzyme in the path￾way, 6-phosphogluconate dehydrogenase (PDH;

EC 1.1.1.44), converts 6-phosphogluconate (6PG) to

ribulose 5-phosphate (RU5P). Loss of the enzyme

activity is lethal, as a high concentration of 6PG is

toxic to eukaryotic cells including Drosophila melano￾gaster [3,4], Saccharomyces cerevisae [5] and Trypano￾soma brucei [2]. 6PG inhibits phosphoglucose iso￾merase [6] and it has been proposed that this disrupts

the main glycolytic pathway, and establishes a positive

feedback loop, although definitive results that identify

the precise mechanism leading to cell death have yet to

be obtained. Extensive kinetic studies have been car￾ried out on PDH [7–11] and crystal structures from

two species have been elucidated [12,13]. The catalytic

conversion of 6PG to RU5P is considered as a three￾step mechanism, with two possible reaction

intermediates (Fig. 1A). Studies using 13C isotope,

Keywords

African trypanosomiasis; enzyme inhibition;

Lactococcus lactis; pentose phosphate

pathway; 6-phosphogluconate

dehydrogenase

Correspondence

W. N. Hunter, Division of Biological

Chemistry and Molecular Microbiology,

School of Life Sciences, University of

Dundee, Dundee DD1 5EH, UK

Fax: +44 1382 385764

Tel: +44 1382 385745

E-mail: [email protected]

(Received 19 September 2006, revised

4 November 2006, accepted 9 November

2006)

doi:10.1111/j.1742-4658.2006.05585.x

Crystal structures of recombinant Lactococcus lactis 6-phosphogluconate

dehydrogenase (LlPDH) in complex with substrate, cofactor, product and

inhibitors have been determined. LlPDH shares significant sequence iden￾tity with the enzymes from sheep liver and the protozoan parasite Trypano￾soma brucei for which structures have been reported. Comparisons indicate

that the key residues in the active site are highly conserved, as are the inter￾actions with the cofactor and the product ribulose 5-phosphate. However,

there are differences in the conformation of the substrate 6-phosphogluco￾nate which may reflect distinct states relevant to catalysis. Analysis of the

complex formed with the potent inhibitor 4-phospho-d-erythronohydroxa￾mic acid, suggests that this molecule does indeed mimic the high-energy

intermediate state that it was designed to. The analysis also identified, as a

contaminant by-product of the inhibitor synthesis, 4-phospho-d-erythrona￾mide, which binds in similar fashion. LlPDH can now serve as a model

system for structure-based inhibitor design targeting the enzyme from

Trypanosoma species.

Abbreviations

PDH, 6-phosphogluconate dehydrogenase; PEA, 4-phospho-D-erythronohydroxamide; PEX, 4-phospho-D-erythronohydroxamic acid; 6PG,

6-phosphogluconate; RU5P, ribulose 5-phosphate.

FEBS Journal 274 (2007) 275–286 ª 2006 The Authors Journal compilation ª 2006 FEBS 275