The three-dimensional structure of the nudix enzyme diadenosine tetraphosphate hydrolase from Lupinus angustifolius L¹

Abstract

The solution structure of diadenosine 5′, 5‴-P¹, P⁴-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap₄A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed β-sheet, a two-stranded antiparallel β-sheet and three helices (αI, αIII, αIV). The root-mean-squared deviation for the backbone (C′, O, N, C^α) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 Å. Several regions, however, show lower definition, particularly an isolated helix (αII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between αII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap₄A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded β-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

Introduction

Diadenosine 5′, 5‴-P¹, P⁴-tetraphosphate (Ap₄A) hydrolase from Lupinus angustifolius L., is an 18.5 kDa member of a subfamily of Ap_nA hydrolases (Dunn et al., 1999), which belong in turn to the Nudix (nucleoside diphosphate linked to x) enzyme family (Bessman et al., 1996). Ap₄A hydrolases have been isolated and characterized from a variety of eukaryote and prokaryote organisms and tissues. Such hydrolases cleave Ap₄A asymmetrically into ATP+AMP, and are thus distinguished from the unrelated and symmetrically cleaving Escherichia coli apaH gene product (Guranowski et al., 1983). Many functions have been suggested for both the enzyme and Ap₄A in cells (McLennan, 1999). Ap₄A is a potential by-product of aminoacyl tRNA synthesis, and accumulation of Ap₄A in cells has been implicated in a range of biological events, including DNA replication, cellular differentiation, heat shock, metabolic stress and even apoptosis (McLennan, 1999). The importance of Ap₄A hydrolases is highlighted by their recently established role in the invasive phenotype of pathogenic bacteria. The gene IalA from Bartonella bacilliformis encodes an asymmetric Ap₄A hydrolase Conyers and Bessman 1999, Cartwright et al 1999. Transformation of E. coli both with this gene and with the IalB gene resulted in an invasive phenotype on otherwise minimally invasive strains, and suggested that asymmetric Ap₄A hydrolase played an important role in invasion by B. bacilliformis(Mitchell & Minnick, 1995). Homology searches suggest that similar Ap₄A hydrolases may be a common feature of many invasive bacteria and a potential target for inhibition of their invasion (Cartwright et al., 1999). The three-dimensional structure of the enzyme and complexes with its substrate or analogues are sought as a prerequisite for design of appropriate inhibitors; however, no structure for the IalA enzyme exists. Ap₄A hydrolase from L. angustifolius, with 35 % sequence identity (McLennan, 1999) is one of the sequences most closely related to IalA (Cartwright et al., 1999)(Figure 1); by comparison, the human analogue has only 18 % identity (McLennan, 1999). Searches of data bases for other Nudix proteins, uncover a family of approximately 300 members, each with a highly conserved Nudix signature: GX₅EX₇REUXEEGU, where U is a hydrophobic residue (Dunn et al., 1999). The only known structure of a member of this family to have been solved is the solution structure of the MutT enzyme, an 8-oxo-guanosine hydrolase from E. coli, in both the free and inhibitor-bound states Frick et al 1995, Lin et al 1996, Lin et al 1997. The fold of the protein includes a central five-stranded β-sheet and two helices, of which one is a catalytic helix that contains part of the Nudix signature. Site-directed mutagenesis, metal titrations and kinetic analysis Lin et al 1997, Harris et al 2000 show that within the Nudix motif are residues essential for catalysis and for the ligation of an enzyme-bound divalent cation. Site-directed mutagenesis of other Nudix hydrolases supports the essential role of these residues Safrany et al 1998, Dunckley and Parker 1999, Yang et al 1999.

Recently, we reported the ¹H, ¹³C and ¹⁵N NMR resonance assignment and secondary structure characterization of the Ap₄A hydrolase from lupin (Swarbrick et al., 2000). These data suggested a structure similar to that of MutT (Lin et al., 1997); however, some differences were noted, including two additional helices and possibly additional β-strands. Here, we describe the 3D solution structure of the metal-free Ap₄A hydrolase from lupin. Further, we describe changes to the ¹H, ¹⁵N spectra of the complex of magnesium and Ap₄A hydrolase on the addition of a diadenosine tetraphosphate analogue, P¹,P⁴-dithio-P²,P³-monochloromethylene diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Chan et al., 1997).