Invariant Ser211 is involved in the catalysis of PD‐L4, type I RIP from Phytolacca dioica leaves

Abstract

Multiple sequence alignment analysis of ribosome inactivating proteins (RIPs) has revealed the occurrence of an invariant seryl residue in proximity of the catalytic tryptophan. The involvement of this seryl residue in the catalytic mechanism of RIPs was investigated by site-directed mutagenesis in PD-L4, type 1 RIP isolated from Phytolacca dioica leaves. We show that the replacement of Ser211 with Ala apparently does not influence the N-beta-glycosidase activity on ribosomes (determined as IC(50) in a cell-free system), but it reduces the adenine polynucleotide glycosylase activity (APG), assayed spectrophotometrically on other substrates such as DNA, rRNA, and poly(A). The ability of PD-L4 to deadenylate polynucleotides appears more sensitive to the Ser211Ala replacement when poly(A) is used as substrate, as only 33% activity is retained by the mutant, while with more complex and heterogeneous substrates such as DNA and rRNA, its APG activity is 73% and 66%, respectively. While the mutated protein shows a conserved secondary structure by CD, it also exhibits a remarkably enhanced tryptophan fluorescence. This indicates that, although the overall protein tridimensional structure is maintained, removal of the hydroxyl group locally affects the environment of a Trp residue. Modelling and docking analyses confirm the interaction between Ser211 and Trp207, which is located within the active site, thus affecting RIP adenine polynucleotide glycosylase activity. Data accumulated so far confirm the potential involvement of Ser211 in the catalytic mechanism of type 1 RIP PD-L4 and a possible role in stabilizing the conformation of Trp207 side chain, which participates actively in the protein enzymatic activity.

Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae)

“…The role of Ser 202 needs to be clarified for its adjacency to the catalytic Trp198 (296 in the sequence logo). We hypothesized that the amino acid could play an important role in stabilizing the conformation of W198 side chain, which was consistent with the report about S211 and W207 in the type 1 RIP PD-L4 [33]. Further analysis showed that SEBIN has the highest similarity with A chains of type II RIPs than any other type I RIPs.…”

Type 1 ribosome-inactivating proteins from Phytolacca dioica L. leaves: differential seasonal and age expression, and cellular localization

“…The following amino acid sequences were used: PAP-, PAP, PAP-I, PAP-II, PAP-R, PAP-S, PAP-S1 and PD-S2 (see "Materials and methods"). For PD-L4 and dioicin 2 from fully expanded leaves of young P. dioica, see Chambery et al (2007) and this work, respectively. All sequences, except dioicin 2, were reported in Entrez database (see "Materials and methods").…”

Structure/function studies on two type 1 ribosome inactivating proteins: Bouganin and lychnin

“…Moreover, it has been proposed that in PD-L4, a type 1 RIP isolated from leaves of Phytolacca dioica, a conserved serine, the fourth residue after the catalytic tryptophan ( Fig. 1), could be also involved into the catalytic mechanism (Chambery et al, 2007;Ruggiero et al, 2008).…”