title: Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme.
abstract: Adenosine 5'-phosphoramidate (NH₂-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH₂-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH₂-pA. The enzyme functions as a homodimer (2 x 15,800 Da). At the optimum pH of 7.0, the K(m) for NH₂-pA was 0.5 µM and k(cat) 0.8 s⁻¹ (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.
authors.affiliation: Department of Biochemistry and Biotechnology, Poznań, University of Life Sciences, Poznań, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Guranowski
authors.forename: Anna M
authors.initials: AM
authors.lastname: Wojdyła
authors.forename: Anna M
authors.initials: AM
authors.lastname: Rydzik
authors.forename: Janusz
authors.initials: J
authors.lastname: Stepiński
authors.forename: Jacek
authors.initials: J
authors.lastname: Jemielity
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D031297:Lupinus / Q000201:enzymology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D010940:Plant Proteins; D000249:Adenosine Monophosphate; D004533:Egtazic Acid; C015651:adenosine 5'-phosphoramidate; D004492:Edetic Acid; D009699:N-Glycosyl Hydrolases