Beschreibung:
<jats:title>ABSTRACT</jats:title>
<jats:p>
NopE1 is a type III-secreted protein of the symbiont
<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Bradyrhizobium japonicum</jats:named-content>
which is expressed in nodules.
<jats:italic>In vitro</jats:italic>
it exhibits self-cleavage in a duplicated domain of unknown function (DUF1521) but only in the presence of calcium. Here we show that either domain is self-sufficient for cleavage. An exchange of the aspartic acid residue at the cleavage site with asparagine prevented cleavage; however, cleavage was still observed with glutamic acid at the same position, indicating that a negative charge at the cleavage site is sufficient. Close to each cleavage site, an EF-hand-like motif is present. A replacement of one of the conserved aspartic acid residues with alanine prevented cleavage at the neighboring site. Except for EDTA, none of several protease inhibitors blocked cleavage, suggesting that a known protease-like mechanism is not involved in the reaction. In line with this, the reaction takes place within a broad pH and temperature range. Interestingly, magnesium, manganese, and several other divalent cations did not induce cleavage, indicating a highly specific calcium-binding site. Based on results obtained by blue-native gel electrophoresis, it is likely that the uncleaved protein forms a dimer and that the fragments of the cleaved protein oligomerize. A database search reveals that the DUF1521 domain is present in proteins encoded by
<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Burkholderia phytofirmans</jats:named-content>
PsNJ (a plant growth-promoting betaproteobacterium) and
<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Vibrio coralliilyticus</jats:named-content>
ATCC BAA450 (a pathogenic gammaproteobacterium). Obviously, this domain is more widespread in proteobacteria, and it might contribute to the interaction with hosts.
</jats:p>