Chambery Angela, Pisante Marianna, Di Maro Antimo, Di Zazzo Erika, Ruvo Menotti, Costantini Susan, Colonna Giovanni, Parente Augusto
Dipartimento di Scienze della Vita, Seconda Università di Napoli, Caserta, Italy.
Proteins. 2007 Apr 1;67(1):209-18. doi: 10.1002/prot.21271.
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.
核糖体失活蛋白（RIPs）的多序列比对分析显示，在催化性色氨酸附近存在一个不变的丝氨酸残基。通过定点诱变对从商陆叶片中分离出的1型RIP——PD-L4中的该丝氨酸残基在RIPs催化机制中的作用进行了研究。我们发现，将Ser211替换为Ala显然不会影响对核糖体的N-β-糖苷酶活性（在无细胞系统中测定为IC50），但会降低对其他底物（如DNA、rRNA和聚腺苷酸）进行分光光度法测定的腺嘌呤多核苷酸糖基化酶活性（APG）。当以聚腺苷酸为底物时，PD-L4使多核苷酸去腺苷酸化的能力对Ser211Ala替换更为敏感，因为突变体仅保留33%的活性，而对于更复杂和异质的底物（如DNA和rRNA），其APG活性分别为73%和66%。虽然突变蛋白通过圆二色谱显示出保守的二级结构，但它也表现出显著增强的色氨酸荧光。这表明，尽管蛋白质的整体三维结构得以维持，但羟基的去除局部影响了一个色氨酸残基的环境。建模和对接分析证实了位于活性位点内的Ser211和Trp207之间的相互作用，从而影响RIP腺嘌呤多核苷酸糖基化酶活性。目前积累的数据证实了Ser211可能参与1型RIP PD-L4的催化机制，并可能在稳定Trp207侧链的构象中发挥作用，而Trp207侧链积极参与蛋白质的酶活性。
