Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease.
The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.
Srivastava
T K
TK
Division of Biopolymers, Central Drug Research Institute, Lucknow, India.
Friedhoff
P
P
Pingoud
A
A
Katti
S B
SB
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry; D006860:Hydrogen Bonding; D013058:Mass Spectrometry; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010755:Organophosphates / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D011061:Poly A / Q000378:metabolism; D012705:Serratia / Q000201:enzymology*; D055672:Static Electricity; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010755:Organophosphates; C021992:dideoxyribonucleoside methylphosphonates; D011061:Poly A; C015465:poly(dA); D004720:Endonucleases; D010727:Phosphoric Diester Hydrolases