Abstract
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. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (Ki = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.
Highlights
C3larvin toxin from P. larvae modifies RhoA protein through its ADP-ribosyltransferase activity
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
The C3 enzymes consist of the Ͻ25-kDa mART domain and have both transferase and GH activity, GH activity is much weaker than transferase activity [8]
Summary
C3larvin toxin from P. larvae modifies RhoA protein through its ADP-ribosyltransferase activity. 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. Tial target residues, mART toxins act on a variety of different proteins within cells, including actin, elongation factor-2, and RhoA [4]. We have solved the crystal structure of C3larvin to 2.30 Å and show that it is a member of the C3 mART subgroup [4] It shows a strong similarity with the C3 toxin subgroup by multiple-sequence alignment, and the likely physiological target is the Rho protein family [8]. The best compounds arising from the screen were tested against C3larvin transferase activity, and one compound was identified as a good lead inhibitor, to our knowledge, the first reported inhibitor of the C3 toxin subclass
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