Stereochemical support for Serratia endonuclease active-site geometry*

Earlier observation that stereoregular All-Rp-PS-oligonucleotides in the presence of nonsequence-specific Serratia endonuclease undergo nucleolytic degradation, confronted with recently published results on the active-site architecture of this enzyme, strongly supports an involvement of 3′-bridging and pro-Sp-nonbridging oxygen atoms of scissile internucleotide bond in the interactions with hydrated magnesium ion anchored by Asn-119 residue of this endonuclease. Recently, the similarity between the active site of I-PpoI specific endonuclease and Serratia marcescens nonsequence-specific endonuclease was pointed out by two independent groups [1,2]. The different folding of these two proteins determines the specificity requirement, but both proteins contain conserved essential asparagine and histidine residues at their active sites and exhibit a similar one metalion co-ordination pattern. Therefore, all main active-site components required for the cleavage of the internucleotide phosphorus–oxygen bond are in the similar arrangement. The general-base histidine 98 (I-PpoI) or 89 (Serratia) residue activates a water molecule for its nucleophilic attack at phosphorus and this occurs opposite from the site of the leaving 3'-oxygen. The ligand for the protein is comprised of the magnesium ion surrounded by three water molecules and Asn-119. Upon binding of the protein, the magnesium ion stabilizes the transition state by coordinating two oxygens of scissile phosphate, namely the 3'-oxygen of P–O–C bond and the pro-Sp-nonbridging oxygen (Fig. 1A; a) [3]. This magnesium ion does not coordinate a water molecule for nucleophilic attack at phosphorus opposite the leaving 3'–oxygen. Such architecture of the active site of the Serratia marcescens endonuclease comprising coordination of two oxygen atoms of the same scissile phosphate to the metal ion seems to be more plausible than the alternate mechanism for the mode of action of Serratia enzyme discussed by Miller et al. [4] and presented in Fig. 1B. The metal-bound water is deprotonated by a general base and attacks the phosphorus atom of the negatively charged phosphate, coordinated to the same metal ion through the 3'-bridging oxygen. Neither of two nonbridging oxygens of phosphate moiety contacts the metal ion. Such a model of active site was also preferred by Flick et al. [5], discussing the mode of action of homing endonuclease I-PpoI. Our choice of the mechanism as presented in Fig. 1A is based upon arguments concerning the stereoselectivity of Serratia enzyme towards stereoregular oligo(nucleoside phosphorothioate)s [6,7]. Since 1995, it has been known that Serratia marcescens endonuclease is stereoselective toward oligo(nucleoside phosphorothioate)s and cleaves only internucleotide phosphorothioates of RP configuration (Fig. 1A; b) [6]. Although that cleavage required 10 higher concentration of enzyme than that effective for the cleavage of isosequental PO-oligonucleotide, a further increase in enzyme concentration does not cause the cleavage of any of eleven phosphorothioate internucleotide bonds of isosequen*Lecture presented at the 13 International Conference on Organic Synthesis (ICOS-13), Warsaw, Poland, 1–5 July 2000. Other presentations are published in this issue, pp. 1577–1797. †Corresponding author -