Characterization of the toxin Plx2A, a RhoA-targeting ADP-ribosyltransferase produced by the honey bee pathogen Paenibacillus larvae.

The toxin Plx2A is an important virulence factor of Paenibacillus larvae, the etiological agent of American Foulbrood, the most destructive bacterial disease of honey bees. Biochemical and functional analyses as well as the crystal structure of Plx2A revealed that it belongs to the C3 mono-ADP-ribosylating toxin subgroup. RhoA was identified as the cellular target of Plx2A activity.

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies.

A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 μm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.

Structural variability of C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADP-ribosyltransferase toxins.

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g.

Characterization of Vis Toxin, a Novel ADP-Ribosyltransferase from Vibrio splendidus.

Vis toxin was identified by a bioinformatics strategy as a putative virulence factor produced by Vibrio splendidus with mono-ADP-ribosyltransferase activity. Vis was purified to homogeneity as a 28 kDa single-domain enzyme and was shown to possess NAD(+)-glycohydrolase [KM(NAD(+)) = 276 ± 12 μM] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [KM(agmatine) = 272 ± 18 mM]. Mass spectrometry analysis revealed that Vis labels l-arginine with ADP-ribose from the NAD(+) substrate at the amino nitrogen of the guanidinium side chain.

C3larvin toxin, an ADP-ribosyltransferase from Paenibacillus larvae.

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD(+) (Km = 34 ± 12 μm) and RhoA (Km = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity.

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand.

Calpains are Ca(2+) dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca(2+) signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy.

Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans.

Distinguishing between calpain heterodimerization and homodimerization.

The two main mammalian calpains, 1 and 2, are heterodimers of a large 80 kDa and a small 28 kDa subunit that together bind multiple calcium ions during enzyme activation. The main contact between the two subunits of these intracellular cysteine proteases is through a pairing of the fifth EF-hand of their C-terminal penta-EF-hand (PEF) domains. From modeling studies and observation of crystal structures, it is not obvious why these calpains form heterodimers with the small subunit rather than homodimers of the large subunit, as suggested for calpain 3 (p94).

Homodimerization of calpain 3 penta-EF-hand domain.

Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass M(r) 80000) and small (M(r) 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution.