Substrate Binding and Catalysis of Ecto-ADP-ribosyltransferase 2.2 from Rat

Abstract

The structures of beta-methylenethiazole-4-carboxamide adenine dinucleotide (TAD), NAD(+), and NADH as bound to ecto-ADP-ribosyltransferase 2.2 from rat and to its mutants E189I and E189A, respectively, have been established. The positions and conformations of NAD(+) and its analogues agree in general with those in other ADP-ribosyltransferases. The kinetic constants for NAD(+) hydrolysis were determined by RP-HPLC. The specific activity amounts to 26 units/mg, which is 6000-fold higher than a previously reported rate and 500-fold higher than the hydrolysis rates of other ADP-ribosyltransferases, confirming that hydrolysis is the major function of this enzyme. On the basis of structures and mutant activities, a catalytic mechanism is proposed. The known auto-ADP-ribosylation of the enzyme at the suggested position R184 is supported by one of the crystal structures where the nucleophile position is occupied by an Neta atom of this arginine which in turn is backed up by the base E159.

Activity and specificity of toxin-related mouse T cell ecto–ADP-ribosyltransferase ART2.2 depends on its association with lipid rafts

“…In ART2.2-Tm, there are 20 amino acids between the hydrophobic transmembrane segment and the fourth conserved cysteine residue (which is engaged in a disulfide bond to the first cysteine in the 3-dimensional structure of rat ART2). 4,38 This corresponds to the distance from the cell membrane in ART2.2-GPI, encompassing the hydrophilic sugar residues of the GPI anchor and 16 amino acids between the site of the GPI anchor attachment on the first Ser residue in the Ser-Gly-Ser motif (, ϩ 1, ϩ 2) and the fourth conserved cysteine (Figure 1). The finding that known target proteins (LFA-1 and P2X7) can undergo ADP-ribosylation by both forms of ART2.2 ( Figure 7) and the finding that both forms of ART2.2 reach similar saturation levels of cell-surface protein ADP-ribosylation at high NAD concentrations and long incubation times indicate that the observed striking differences in activity of ART2.2-GPI compared with ART2.2-Tm at low substrate concentrations and short incubation times ( Figure 5) are not caused by differences in the lengths of the juxtamembrane stalk.…”

Characterization of a Unique ADP-Ribosyltransferase of Mycoplasma penetrans

“…It was previously reported that the conserved polar glutamine/glutamate (Q/E) residue located two amino acids upstream of the catalytic glutamate in ADPRTs may determine substrate specificity (15). The substitution of this glutamate with the polar lysine residue in MYPE9110 may account for distinctions between CARDS toxin and MYPE9110, as alterations of targets have been linked to changes in this amino acid in eukaryotic ADPRTs (30). Both of these mycoplasma toxins were able to hydrolyze NAD in the absence of target proteins, although the Clostridium C3 and M. pneumoniae CARDS toxins exhibited higher levels of activity, which could be due to the absence of the STS motif in MYPE9110.…”

“…PCR amplification of the M. penetrans tufA gene (MYPE320) was performed with primers corresponding to the internal region of nucleotides 505 to 906 of tufA by using EF-Tu FP (5Ј-ATGGCAAAACAAAA GTTTGATAGATCAAAAG-3Ј) and EF-Tu RP (5Ј-TTATCTAATAACTTTA GTTACTGTACCAGC-3Ј). To determine whether MYPE9110 was secreted during M. penetrans growth, supernatants of exponentially grown cultures were ammonium sulfate precipitated (30,50, and 80% saturations). Each fraction was desalted, concentrated, and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels.…”

Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto–ADP-Ribosyltransferase ARTC2.2

“…8. Note that the proteolytic cleavage site occurs 13 aa downstream of C226, which in turn is engaged in an intrachain disulfide bond conserved in the ARTC family (49,50). The amino acids between C226 and the GPI anchor presumably form a flexible stalklike structure.…”