Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism

Abstract

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

Keywords: ATP hydrolysis; Dolichos biflorus; NTPDase; Trifolium repens; Vigna unguiculata subsp. cylindrica; apyrase.

Figure 1

Amino acid sequence alignment of structurally similar homologues for 7WC and DbLNP plant NTPDases and the hydrolysis catalytic mechanism. (a) Apyrase conserved regions (ACRs; purple) are numbered 1 through 5. Cysteine pairs for disulphide bonds (yellow) are shown as coloured boxes. Asterisks (blue) denote the residues involved in the catalytic site and/or mechanism. 3ZX0 and 4CD1 are NTPDase I and II, respectively, from the species R. novegicus, and 4BVO is NTPDaseI from L. pneumophila. (b) Catalytic residues are labelled according to DbLNP residue numbering and represent ACRs 1, 3, 4, and 5. ACR2 resides involved in the active site are omitted for clarity.

Figure 2

The crystal structures of 7WC‐apo (a) and DbLNP‐PO4 (b) NTPDases and their apyrase conserved regions (ACRs) (c) and (d) for 7WC and DbLNP, respectively. Domain 1 and domain 2 are colored green and blue, respectively, disulphide bonds colored orange, phosphates shown as red sticks and manganese ion as a pink sphere. ACR1, −2, −3, −4, and −5 represented as cyan, hot pink, green, purple, and marine in color, respectively. An interactive view is available in the electronic version of the article

Figure 3

Active site of 7WC and DbLNP showing residues involved in binding. (a) 7WC‐apo‐2 phosphate ions in the active site (red sticks), (b) 7WC‐AMP‐AMP in the active site (red and blue sticks), (c) 7WC‐adenine‐1 adenine molecule (blue and orange sticks) and 2 phosphate ions (red sticks) in the active site, (d) DbLNP‐PO4–2 phosphate ions (red sticks) and a manganese ion (purple sphere) in the active site. Red spheres represent water molecules. (e) Superposition of 7WC‐apo (green) and 7WC‐AMP (purple) including phosphate and AMP molecules and (f) superposition of 7WC‐apo (green) and 7WC adenine (yellow) including phosphate and adenine molecule. An interactive view is available in the electronic version of the article

Figure 4

Cartoon representation of the most closely related NTPDase PDB structures individually superimposed by secondary structure with 7WC‐apo (green) and DbLNP‐PO4 (blue). (a) 7WC with R. novegicus NTPDase1 (3ZX0; cyan), (b) 7WC with L. pneumophila NTPDase1 (4BVO; pink), (c) 7WC with N. caninum NTPDase (3AGR; orange), (d) DbLNP with R. novegicus NTPDase1 (3ZX0; cyan), (e) DbLNP with L. pneumophila NTPDase1 (4BVO; pink) and (f) DbLNP with N. caninum NTPDase (3AGR; orange). An interactive view is available in the electronic version of the article