R tag directed us to examine the role of the TAT signal sequence in YedY expression. The TAT machinery allows protein translocation to the periplasm, in a fully folded form with inserted cofactors. Several studies have described the existence of a “control” exerted to avoid translocation of unfolded proteins that are consequently degraded [31]. When R. sphaeroides YedY is expressed in E. coli in the absence of signal sequence, the protein accumulates in an insoluble and inactive form (Figure 4). This could be due to the nonphysiologic growth conditions used (overnight incubation at 16 ). However, using these same growth conditions, in the presence of the signal sequence, does not result in any inclusion bodies, and the protein is synthesized in an active form. This demonstrates that the presence of the signal sequence is important to stabilize the enzyme. Even though the signal sequence differs between E. coli YedY and R. sphaeroides YedY (22 identity), the R. sphaeroides signal sequence is necessary for heterologous expression in E. coli. Hilton et al. [18] obtained a different result for heterologous expression of R. sphaeroides DMSO reductase. In their study, they showed (by zymogram and western blot) that the enzyme was synthesized in a high amount in the presence of the signal sequence, even though it was inactive in E. coli. By contrast, enzyme synthesis was considerably reduced in the absence of the signal sequence, although an active enzyme was still produced. This shows that the role of the signal sequence in the maturation process can differ from one enzyme to another and is speciesdependent. For example, R. sphaeroides is able to produce a fully active YedY (Figures 6 and 7) in the absence of its signal sequence, as opposed to E. coli, but with a very low yield (approximately 10-fold less than with the signal sequence). This illustrates that the signal sequence is not strictly required for the insertion of the molybdenum cofactor and enzyme folding; on the other hand, it provides critical help in YedY biogenesis, most probably via the involvement of Velpatasvir site chaperones that protect apoenzyme from proteolysis. Indeed, the REMP (Redox Enzyme Maturation Protein) group of chaperones are involved in maturation and insertion of cofactors for TATdependent redox enzymes [32]. They are often found in the same operon as their corresponding gene-encoded TAT substrates. The best characterized ones are TorD and DmsD from E. coli; these interact with the signal sequence of their substrate (TorA and DmsA, respectively) as well as with other enzymes [32,33]. Although these chaperones are crucial to obtain a high amount of fully active molybdoenzymes, some active enzyme is still produced in their absence [15]. This is probably whatSabaty et al. BMC Biochemistry 2013, 14:28 http://www.biomedcentral.com/1471-2091/14/Page 9 ofhappens when YedY is expressed without its signal sequence: the interaction with putative REMP may be impaired, but a small amount of active enzyme is still produced. No gene encoding a putative specific chaperone has been found in the genomic region of yedYZ. It could thus be localized elsewhere in the genome, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 or YedY maturation could involve some other well-known REMP. Recently, it was shown in E.coli that neither DmsD nor TorD are necessary for YedY maturation [34]. Mechanisms and chaperones involved in TAT-dependent translocation for R. sphaeroides have not yet been described, and it remains unknown if the maturation of YedY in.
