4972 Nucleic Acids Research, 2012, Vol. 40, No. 11 not import Asn (44), and given the fact that no Asn synthetase or AsnRS could be detected in the genome using BLAST, the indirect misacylation/transamidation pathway seems to be the sole route for Asn and Asn-tRNAAsn biosynthesis, as is also the case for Gln-tRNAGln (24). Hp ND-AspRS and GatCAB partici- pate together with tRNAAsn in the formation of a ribonucleoprotein homologous to that shown for T. thermophilus (Figures 1 and 2). Contrary to the T. thermophilus Asn-transamidosome (21), we noticed that, in the Hp system, uncharged tRNAAsn has a 10-fold stronger afﬁnity for GatCAB (the second enzyme of the pathway) than for ND-AspRS (the ﬁrst enzyme in the pathway) (Figures 1 and 2). Thus, in H. pylori, ND-AspRS presumably docks onto the tRNPC, rather than to free tRNAAsn (Scheme 1). In conditions where GatCAB is in excess, tRNAAsn may thus be mostly pre- sented by GatCAB, ensuring the proper channeling of Asp-tRNAAsn toward GatCAB. Consistently, the tRNPC seems to be a better substrate for aminoacylation than free tRNAAsn (Table 1 and Figure 4). Upon aspartylation, the Asp-tRNAAsn acceptor-end shifts toward the GatCAB active site and triggers the release of ND-AspRS from the complex (Figure 2, lanes 7–9), explaining why the steady-state rate of ND-AspRS in- creases in the transamidosome. Indeed, when GatCAB is present, Asp-tRNAAsn gets pulled out of the ND-AspRS active site, freeing ND-AspRS from its product and allowing a new catalytic cycle to occur as evidenced by the faster steady-state rate of aspartylation. This scheme strongly differs from that determined for the T. thermophilus Asn-transamidosome, where the complex remains stable over the entire catalytic cycle, according to a peculiar ﬂip-ﬂop mechanism involving a non-substrate scaffold tRNA (scaftRNA) (22). Indeed, Tt ND-AspRS, unlike Hp ND-AspRS, changes its release mode and retains Asp-tRNAAsn, to preserve the tRNP stability when GatCAB is present, e.g. within the complex. These major differences in stability and in mech- anism could be due to the differences between Tt and Hp ND-AspRSs. Indeed, while Tt ND-AspRS belongs to the archaeal structural-type, Hp ND-AspRS falls into the bacterial structural-type which contains an additional domain, called the GAD domain. This domain is appended to the active site where it could prevent the scaffold tRNA from playing its role, or it could destabilize the overall complex (22). Either of these effects would result in lesser stability and greater dynamics for the bacterial-type Asn-transamidosome. On the other hand, while Tt ND-AspRS does not retain its Asp-tRNAAsn product in the absence of GatCAB (22) (i.e. outside the complex), Hp ND-AspRS might be able to trap this aminoacyl-tRNA thanks to the GAD domain, which lies next to the active site in a position where an editing domain is found in the class II homologous ThrRS (45) ND-AspRS displays a dual mode of release for its two Asp-tRNA products ND-AspRS recognizes and aspartylates both tRNAAsp and tRNAAsn equivalently, which makes it a ND enzyme for substrate recognition and aminoacylation. Nevertheless, kinetic studies clearly show a difference in product release. Asp-tRNAAsn is retained upon aminoacylation, while Asp-tRNAAsp is not (Figure 3). This difference seems to be triggered by the overall con- formation of tRNA rather than by speciﬁc identity elements. Our results suggest that the shapes of tRNAAsp and tRNAAsn could induce different active site conﬁgurations that lead to these divergent modes of release. According to the predominant effect of pH on the steady-state rate (slower phase) (Supplementary Figure S1), the CCA-Asp moiety may play an important role in Asp-tRNAAsn retention on ND-AspRS. This is further conﬁrmed with differential inhibition patterns using Asp-ol-AMP (Supplementary Table S1) in presence of tRNAAsp or tRNAAsn, the latter tRNA pro- tecting the active site from inhibition more efﬁciently as a consequence of its retention. This is in agreement with a previous work on P. aeruginosa ND-AspRS (39) showing that the limiting step for Asp-tRNAAsn synthesis could be general for ND-AspRS of bacterial type. This scheme strongly shares analogy with editing mech- anisms, where a tRNA gets misacylated with a wrong or near-cognate amino acid and retained on the enzyme to be edited. However, here, a cognate amino acid is loaded onto a ‘non-cognate’ tRNAAsn and the Asp-tRNAAsn product gets retained, perhaps to statistically favor further processing of the misacylated intermediate by GatCAB prior to its release (Scheme 1). We named this a ‘rescue mechanism’; it may be essential under conditions where GatCAB is not found in sufﬁcient amounts within the cell or when it is occupied in the glutaminylation pathway that also exists in H. pylori. Taking into account the hypothesis forged from a kinetic study (8), the rate-limiting step of ND-AspRS, like all class II aaRSs, is expected to be the transfer of activated Asp onto tRNA. In contrast, in class I aaRSs, the rate-limiting step is typically product release. Clearly, Hp ND-AspRS shifts to a class I-like kinetic behavior with tRNAAsn but retains its class II character with tRNAAsp. Previously, it has been demonstrated that the class II phosphoseryl-tRNA synthetase (SepRS) used in the Cys-tRNACys biosynthesis indirect pathway in Methanocaldococcus janaschii, also has a rate-limiting step for the release of the aminoacyl-tRNA (46). The authors hypothesized that this characteristic could have evolved from modiﬁcations of the motifs implicated in aminoacylation, accelerating the transfer step over release, leading to a new rate-limiting step compared to other class II enzymes that is more similar to class I. ND-AspRS is a new example of an aaRS that can shift from a class II to a class I-like kinetic behavior, according to its tRNA substrate. This phenomenon fully correlates with the fates of Asp-tRNA products, since Asp-tRNAAsn should be retained on ND-AspRS until GatCAB transforms it into Asn-tRNAAsn, while Asp-tRNAAsp should be released in line with its ‘cognate’ status. Together, these results allow us to make an analogy between the misacylation/transamidation pathway and editing strategies encountered with several classical aaRSs.