title: RCL hydrolyzes 2'-deoxyribonucleoside 5'-monophosphate via formation of a reaction intermediate.
abstract: RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2'-deoxyribonucleoside 5'-monophosphates. Recently, the structures of both free wild type and GMP-bound mutant complex have been determined by multidimensional NMR, revealing a doubly wound α/β protein existing in a symmetric homodimer. In this work, we investigated the catalytic mechanism by rational site-directed mutagenesis, steady-state and pre-steady-state kinetics, ITC binding analysis, methanolysis, and NMR study. First, we provide kinetic evidence in support of the structural studies that RCL functions in a dimeric form, with an apparent dissociation constant around 0.5 μM in the presence of substrate dGMP. Second, among the eight residues under investigation, the highly conserved Glu93 is absolutely critical and Tyr13 is also important likely contributing to the chemical step, whereas Ser117 from the neighboring subunit and Ser87 could be the key residues for the phosphate group recognition. Lastly, we demonstrate by methanolysis study that the catalytic reaction proceeds via the formation of a reaction intermediate, which is subsequently hydrolyzed by solvent nucleophile resulting in the formation of normal product deoxyribose monophosphate (dR5P) or methoylated-dR5P. In conclusion, the current study provides mechanistic insights into a new class of nucleotide hydrolase, which resembles nucleoside 2'-deoxyribosyltransferases structurally and functionally but also possesses clear distinction.
authors.affiliation: Biochemistry Department, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Doddapaneni
authors.forename: Walter
authors.initials: W
authors.lastname: Zahurancik
authors.forename: Adam
authors.initials: A
authors.lastname: Haushalter
authors.forename: Chunhua
authors.initials: C
authors.lastname: Yuan
authors.forename: Jane
authors.initials: J
authors.lastname: Jackman
authors.forename: Zhengrong
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D055162:Biocatalysis; D003854:Deoxyribonucleotides / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
chemical_list: D003854:Deoxyribonucleotides; C518894:DNPH1 protein, rat; D009699:N-Glycosyl Hydrolases