RCL hydrolyzes 2'-deoxyribonucleoside 5'-monophosphate via formation of a reaction intermediate.

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/ß 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.

Solution structure of RCL, a novel 2'-deoxyribonucleoside 5'-monophosphate N-glycosidase.

RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2'-deoxyribonucleoside 5'-monophosphates, a novel enzymatic reaction reported only recently. In this work, we determined the solution structure by multidimensional NMR and provide a structural framework to elucidate its mechanism with computational simulation. RCL is a symmetric homodimer, with each monomer consisting of a five-stranded parallel beta-sheet sandwiched between five alpha-helices. Three of the helices form the dimer interface, allowing two monomers to pack side by side. The overall architecture featuring a Rossmann fold is topologically similar to that of deoxyribosyltransferases, with major differences observed in the putative substrate binding pocket and the C-terminal tail. The latter is seemingly flexible and projecting away from the core structure in RCL, but loops back and is positioned at the bottom of the neighboring active site in the transferases. This difference may bear functional implications in the context of nucleobase recognition involving the C-terminal carboxyl group, which is only required in the reverse reaction by the transferases. It was also noticed that residues around the putative active site show significant conformational variation, suggesting that protein dynamics may play an important role in the enzymatic function of apo-RCL. Overall, the work provides invaluable insight into the mechanism of a novel N-glycosidase from the structural point of view, which in turn will allow rational anti-tumor and anti-angiogenesis drug design.