ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli. The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives. IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5-tetraphospho-5-adenosine (Ap 4 A), but also hydrolyzing Ap 5 A, Ap 6 A, Gp 4 G, and Gp 5 G. In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety.Bartonella bacilliformis is the only bacterium known to invade human red blood cells, and it and other species of Bartonella are responsible for several maladies, including Carrion's disease (Oroya fever, verruga peruana), cat scratch disease, trench fever, bacilliary angiomatosis, and bacilliary endocarditis (1, 2). In their studies on the invasiveness of B. bacilliformis, Mitchell and Minnick identified a two-gene locus which, when transformed into minimally invasive Escherichia coli, markedly increased their capacity to invade red blood cells in vitro (3). This report attracted our attention, because one of these two genes, ialA (for invasion-associated locus) codes for an open reading frame containing a small array of highly conserved amino acids we have called the Nudix box (4) (see Sequence 1).All 16 of the enzymes containing this Nudix signature sequence, discovered so far (see Ref. 5), catalyze the hydrolysis of nucleoside diphosphates linked to some other moiety, X, hence the acronym (see "Note Added in Proof "). The Nudix box is represented in all three kingdoms, Archaea, Prokaryota, and Eukaryota, from viruses to humans. Recent BLAST (6) searches have uncovered over 200 putative proteins containing the Nudix motif in 60 species, and we are systematically studying the members of this primordial and widely distributed family.This communication describes the cloning of ialA, and the expression, purification, and partial characterization of the invasion-associated protein, IalA, as an enzyme catalyzing the hydrolysis of dinucleoside 5Ј-polyphosphates. EXPERIMENTAL PROCEDURES MaterialsAll biochemicals were from Sigma. Enzymes used in standard cloning procedures were from Life Technologies Inc., Stratagene, or U. S. Biochemical Corp., except the Pfu DNA polymerase from Perkin-Elmer. Oligonucleotide primers were from Integrated DNA Technologies. E. coli HMS174(DE3) cells and pET11b were obtained from Novagen. E. coli HB101 cells were from our laboratory stock. pGroESL, from George H. Lorimer, DuPont, contained the groEL and groES genes, a T7 lac promoter, and a chloramphenicol resistance gene. Sephadex G-100 was from Amersham Pharmacia Biotech. MethodsCloning-The ialA gene (GenBank TM accession number L25276) from B. bacilliformis...

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.

Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5-GGCGGCGGT/TGGGGCTCG-3 [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.Adeno-associated virus serotype 2 (AAV-2) is a human parvovirus. AAV-2 normally requires a helper virus, such as an adenovirus or herpesvirus for productive infection (7). In the absence of helper virus, AAV-2 can establish a latent infection by integrating its genome into the host DNA. AAV-2 preferentially integrates its DNA into a 4-kb region of human chromosome 19 (19q13-qter), designated AAVS1 (27-30). Subsequent infection by helper virus leads to rescue and productive infection. Preferential integration into AAVS1 requires the Rep68 or Rep78 protein (Rep68/78), encoded by AAV-2, as well as specific DNA sequences within the virus genome and AAVS1 (3,11,35,42,47,55,57,59). Binding sites for Rep68/78 within both the viral DNA and AAVS1 appear to be required (35,42,55,59). These sites, called Rep recognition sequences (RRSs), are comprised of imperfect repeats of the sequence 5Ј-GCTC-3Ј or its complement, 5Ј-GAGC-3Ј (11,20,59). Rep68/78 can form a bridge between RRS-containing DNAs that may facilitate preferential integration (34, 59).Rep68 and Rep78 also have a nucleoside triphosphate-dependent, strand-specific, site-specific endonuclease (nicking) activity (22,23). A Rep68/78 nicking site is called a terminal resolution site (trs) because of the role of such nicking sites in AAV-2 replication (22, 50, 52). Nicking requires both a specific sequence flanking the trs and a nearby...